Pulmonary administration of chemically modified insulin

ABSTRACT

The present invention provides active, hydrophilic polymer-modified derivatives of insulin. The insulin derivatives of the invention are, in one aspect, suitable for delivery to the lung and exhibit pharmakokinetic and/or pharmacodynamic properties that are significantly improved over native insulin.

[0001] This application claims the benefit of priority of U.S.Provisional patent application Ser. No. 60/292,423, the content of whichis incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention is directed to bioactive, hydrophilicpolymer-modified insulin derivatives for delivery to the lung byinhalation. Methods for preparing and administering such derivatives arealso provided.

BACKGROUND OF THE INVENTION

[0003] Insulin is a polypeptide hormone that is produced in thepancreatic β-cells of normal (non-diabetic) individuals. Human insulinis a 51 amino acid polypeptide hormone with a molecular weight of about5800 daltons. The insulin molecule is composed of two peptide chains (anA and a B chain) containing one intrasubunit and two intersubunitdisulfide bonds. The A chain is composed of 21 amino acids while the Bchain is composed of 30 amino acids. The two chains of insulin form ahighly ordered structure with several α-helical regions in both the Aand the B chains. Interestingly, the isolated chains of insulin areinactive. In solution, insulin can exist as a monomer or as a dimer oras a hexamer. Insulin is hexameric in the highly concentratedpreparations used for subcutaneous therapy but becomes monomeric as itis diluted in body fluids. Insulin is necessary for regulatingcarbohydrate metabolism by reducing blood glucose levels; a systemicdeficiency of insulin causes diabetes. The survival of diabetic patientsdepends on the frequent and long-term administration of insulin tomaintain acceptable blood glucose levels.

[0004] Current insulin formulations possess deficiencies that can leadto serious medical complications in the treatment of diabetes. Forinstance, the standard zinc insulin preparation most commonly used bydiabetics exists as a suspension of microcrystals of inactive hexamericinsulin. Dissolution of the microcrystals followed by dissociation ofthe hexamer into the active monomer form can lead to delayed andindividually variable absorption of insulin into the bloodstream (F.Liu, et al., Bioconjugate Chem., 8, 664-672 (1997); T. Uchio, et al.,Adv. Drug Del. Rev., 35, 289-306 (1999); K. Hinds, et al., BioconjugateChem., 11, 195-201 (2000). Formulations of insulin also suffer fromphysical instability due to the tendency of insulin to form fibrils andinsoluble precipitates. Precipitation is especially problematic forformulations intended for use in insulin pumps. Formulated insulin isalso prone to chemical degradation, e.g., non-enzymatic deamidation andformation of high molecular weight transformation products such ascovalent insulin dimers (Brange, J., et al., Pharm. Res., 9, 715-726(1992); Brange, J., et al., Pharm. Res., 9, 727-734 (1992). There issignificant evidence that the incidence of immunological responses toinsulin may result from the presence of these covalent aggregates ofinsulin (Robbins, D. C., et al., Diabetes, 36, 838-841 (1987). Moreover,even highly purified human insulin is slightly immunogenic. (Kim, ibid.)

[0005] Apart from the formulation instability problems noted above,there are also numerous drawbacks associated with current insulintherapies from an administration standpoint. Insulin is most commonlyadministered by subcutaneous injection, typically into the abdomen orupper thighs. Insulin may also be administered intravenously orintramuscularly. In order to maintain acceptable blood glucose levels,it is often necessary to inject insulin at least once or twice per day,with supplemental injections of insulin being administered whennecessary. Aggressive treatment of diabetes can require even morefrequent injections, where the patient closely monitors blood glucoselevels using a home diagnostic kit. The administration of insulin byinjection is undesirable in a number of respects. First, many patientsfind it difficult and burdensome to inject themselves as frequently asnecessary to maintain acceptable blood glucose levels. In fact, manyType 2 patients avoid going on insulin for years because of needlephobia. Such reluctance can lead to non-compliance, which in the mostserious cases can be life-threatening. Moreover, systemic absorption ofinsulin from subcutaneous injection is relatively slow, frequentlyrequiring from 45 to 90 minutes, even when fast-acting insulinformulations are employed. Thus, it has long been a goal to providealternative insulin formulations and routes of administration whichavoid the need for self-injection and which can provide rapid systemicavailability of insulin.

[0006] Numerous non-injectable formulation types such as oral and nasalhave been explored, however, no commercially viable oral or nasal-baseddelivery system for insulin has been developed as a result of theseefforts (Patton, et al., Adv. Drug Delivery Reviews, 1, 35 (2-3),235-247 (1999)), mainly due to very low and variable bioavailability(Hilsted, J., et al., Diabetologia 38, 680-684, (1995)). Althoughbioavailability can be increased with absorption enhancers, these agentscan damage the mucosa.

[0007] However, inhaleable formulations of insulin have been developedwhich appear to be quite promising in overcoming many of the problemsnoted above. For example, U.S. Pat. No. 5,997,848 (Patton, et al.,Inhale Therapeutic Systems, Inc.) describes dry powder formulations ofinsulin which (i) are chemically and physically stable at roomtemperature, and (ii) when inhaled, are rapidly absorbed through theepithelial cells of the alveolar region into the blood circulation. Therapid-acting insulin formulations and methods described therein avoidthe need for burdensome self-injections, and have been shown in threemonth human efficacy studies to provide equivalent glucose control inType I and Type II insulin-dependent diabetics when compared tosubcutaneous injection (Patton, et al., Adv. Drug Delivery Reviews, 1,35 (2-3), 235-247 (1999)). The dry powder insulin formulations describedby Patton, et al., while overcoming the problems of formulationinstability and patient non-compliance, still require frequent (e.g.,mealtime) inhalations of insulin for effective control of glucoselevels. Moreover, a typical insulin dosing regime of this type, based onrapid acting inhaleable insulin, still requires a single injection oflong-acting insulin at bedtime for Type I and some Type II diabetics.Thus, there still exists a need for active, soluble, stable forms ofinsulin that require less frequent dosing, i.e., long-acting insulinformulations, preferably administrable by inhalation.

[0008] Long-acting insulin formulations are ideally characterized ashaving a very slow onset and a prolonged, relatively flat peak ofaction. Current long acting injectable insulin formulations, e.g.,ultralente (extended insulin zinc suspension) and protamine zinc insulinsuspension, are very unsatisfactory. These formulations tend to peakrather than provide a low basal concentration of insulin, areunpredictable, and typically exhibit a duration of action of no longerthan about a day. The long half-life of ultralente insulin makes itdifficult to determine the optimal dosage range, and protamine zincinsulin is rarely used because of its unpredictable and prolonged courseof action (Goodman & Gilman, “The Pharmacological Basis of Therapeutics,Ninth Ed., Hardman and Limbird, eds, 1996, p. 1500). Other long-actinginjectable formulations which have been explored unsuccessfully includealbumin-bound insulin and cobalt-insulin hexamer formulations (Hoffman,A., Ziv E., Clin. Pharmacokinet, 33(4):285-301 (1997)).

[0009] A number of long-acting pulmonary insulin formulations have alsobeen explored. These include liposomes containing a large excess oflipid relative to insulin (Liu. F-Y, et al., Pharm. Res. 10, 228-232,(1993)), porous poly(lactic acid-co-glycolic acid) (PLGA) insulinparticles (Edwards, D. A., et al., Science 276(5320), 1868-1871 (1997)),nebulized PLGA nanospheres (Kawashima, Y., et al., J. ControlledRelease, 62(1-2): 279-287 (1999)) and phospholipid/protamine insulinformulations (Vanbever, R., et al., Proc. Control Rel. Bioact. Mater.25, 261-262 (1998)). Unfortunately, all of these formulations haveproven unsatisfactory, due to either low bioavailabilities whenadministered in rats, or due to formulation insufficiencies. Thus, along-felt need exists for optimized long-acting insulin formulationsthat are bioactive, physically and chemically stable, water-soluble, andpreferably monomeric. Ideally, such formulations will preferably besuited for pulmonary administration.

SUMMARY OF THE INVENTION

[0010] In one aspect, the present invention is based upon compositionsof insulin for administration to the systemic circulation via the deeplung. Specifically, the compositions of the invention comprise aconjugate of insulin covalently coupled to one or more molecules of anon-naturally occurring hydrophilic polymer. In a preferred embodiment,the non-naturally occurring, hydrophilic polymer covalently coupled toinsulin is a polyalkylene glycol such as polyethylene glycol (PEG),although all of the embodiments set forth herein may be equally appliedto other non-naturally occurring hydrophilic polymers.

[0011] In general, an insulin-polymer conjugate of the invention willexhibit pharmacokinetic and pharmacodynamic properties improved overnative insulin, particularly when administered to the lung. In oneembodiment, the PEG-insulin conjugates provided herein exhibit goodabsolute bioavailabilities when administered to the lung and deep lung.In a particular embodiment, a PEG-insulin conjugate of the invention ischaracterized by an absolute pulmonary bioavailability that is greaterthan that of native insulin. Preferably, a PEG-insulin conjugate of theinvention is characterized by having an absolute pulmonarybioavailability that is at least 1.5-2.0 times greater than that ofnative insulin. In a more preferred embodiment, a PEG-insulin conjugatein accordance with the invention is characterized by an absolutepulmonary bioavailability that is greater than about 15%, even morepreferably greater than about 20% or most preferably greater than about30%.

[0012] In yet another embodiment, a PEG-insulin conjugate of theinvention, when administered pulmonarily, exhibits a Tmax (time requiredto reach maximum concentration) that is at least 1.5 times that ofnative insulin, or more preferably is at least 2 or 3 times, or evenmore preferably that is at least five times that of native insulin.

[0013] PEGs for use in the conjugates of the invention may possessseveral different features. In one embodiment of the invention, thepolyethylene glycol-portion of a PEG-insulin conjugate as describedherein is end-capped with an inert or non-reactive terminal group suchas an alkoxy group or more specifically methoxy group.

[0014] In an alternative embodiment, the polyethylene glycol portion ofthe conjugate will possess an architecture particularly well suited forattachment to insulin including linear polyethylene glycols andmulti-armed or branched polyethylene glycols. In yet another embodiment,a PEG-insulin conjugate may comprise two mono-functionally-derivatizedinsulin molecules interconnected by a di-activated polyethylene glycol(insulin-PEG-insulin). Alternatively, an insulin molecule within this“dumbell” architecture may be further modified by additional PEGs.

[0015] In another embodiment, a PEG-insulin conjugate of the inventioncomprises a forked polyethylene glycol having a branching moiety at oneend of the polymer chain and two free reactive groups (or a multiple oftwo) linked to the branching moiety for covalent attachment to insulin.In this embodiment of the invention, the branched architecture ofpolyethylene glycol allows attachment of the polymer chain to two ormore molecules of insulin.

[0016] The polyethylene glycol-portion of an insulin conjugate of theinvention may optionally contain one or more degradable linkages.

[0017] Typically, insulin is covalently coupled to PEG via a linkingmoiety positioned at a terminus of the PEG. Preferred linking moietiesfor use in the invention include those suitable for coupling withreactive insulin amino groups such as N-hydroxysuccinimide activeesters, active carbonates, aldehydes, and acetals.

[0018] In yet another embodiment, a PEG covalently coupled to insulin ina conjugate of the invention will comprise from about 2 to about 300subunits of (OCH₂CH₂), preferably from about 4 to 200 subunits, and morepreferably from about 10 to 100 subunits.

[0019] In an alternative embodiment, a PEG covalently coupled to insulinwill possess a nominal average molecular weight of from about 200 toabout 10,000 daltons. In a preferred embodiment, the PEG will possess anominal average molecular weight from about 200 to about 5000 daltons.In yet a more preferred embodiment, the PEG will possess a nominalaverage molecular weight from about 200 to about 2000 daltons or fromabout 200 to about 1000 daltons.

[0020] In a particular embodiment, the insulin portion of the conjugatecomprises native human insulin.

[0021] In yet another embodiment, the conjugate of the composition ofthe invention possesses a purity of greater than about 90% (i.e., of theconjugate portion of the composition, 90% or more by weight comprisesone or more PEG-insulins). That is to say, compositions of the inventionmay be characterized by a high degree of purity of conjugated insulincomponent, i.e., the composition is absent detectable amounts of freepolyethylene glycol species and other PEG-related impurities.

[0022] In one embodiment, a composition of the invention comprises aconjugate wherein insulin is covalently coupled to PEG at one or more ofits amino sites. Insulin contained within a composition of the inventionmay be mono-substitituted (i.e., having only one PEG covalently coupledthereto). Particular mono-substituted PEG-insulin conjugates inaccordance with the invention possess a polyethylene glycol moietycovalently attached to a position on the insulin molecule selected fromthe group consisting of PheB1, GlyA1 and LysB29.

[0023] In a preferred embodiment, the PEG moiety is covalently attachedat the PheB 1 site of insulin. In a one embodiment, at least about 75%of the B-1Phe sites on insulin are covalently coupled to PEG. In anotherembodiment, at least about 90% of the B-1 Phe sites on insulin arecovalently coupled to PEG.

[0024] Compositions of the invention may also comprise a mixture ofmono-conjugated and di-conjugated PEG insulin having any one or more ofthe features described above. Such compositions may further comprise atri-conjugated PEG insulin.

[0025] In an alternative embodiment, a PEG insulin conjugate inaccordance with the invention is characterized by a rate of proteolysisthat is reduced relative to non-pegylated or native insulin.

[0026] A composition according to the invention may also comprise amixture of a PEG-insulin conjugate and non-chemically modified or nativeinsulin.

[0027] Also encompassed is a composition as described above inaerosolized form.

[0028] Compositions of the invention may be dissolved or suspended inliquid or in dry form, and may additionally comprise a pharmaceuticallyacceptable excipient.

[0029] Also provided herein is a bioactive polyethylene glycol-insulinconjugate suitable for administration by inhalation to the deep lung.

[0030] In yet another aspect, the invention provides a method fordelivering a PEG-insulin conjugate to a mammalian subject in needthereof by administering by inhalation a PEG-insulin composition aspreviously described in aerosolized form.

[0031] The invention also provides in another aspect, a method forproviding a substantially non-immunogenic insulin composition foradministration to the lung. The method includes the steps of covalentlycoupling insulin to one or more molecules of a non-naturally occurringhydrophilic polymer conjugate as described herein, and administering thecomposition to the lung of subject by inhalation, whereby as a result,the insulin passes through the lung and enters into the bloodcirculation.

[0032] In another aspect, provided is a method for providing a prolongedeffect insulin composition for administration to the lung of a humansubject. The method includes covalently coupling insulin to one or moremolecules of a non-naturally occurring hydrophilic polymer to provide acomposition that includes an insulin-hydrophilic polymer conjugate, andadministering the composition to the lung of the subject by inhalation.Upon the administering step, insulin passes through the lung and entersinto the blood circulation and elevated blood levels of insulin aresustained for at least 8 hours post administration.

[0033] A PEG-insulin conjugate of the invention, when aerosolized andadministered via inhalation, is useful in the treatment of diabetesmelliltus (DM).

[0034] These and other objects and features of the invention will becomemore fully apparent when the following detailed description is read.

BRIEF DESCRIPTION OF THE FIGURES

[0035]FIG. 1 is a plot of the rate of enzymatic digestion of anillustrative PEG-insulin conjugate (“750-2 PEG insulin”) versus anunmodified insulin control as described in detail in Example 6.

[0036]FIG. 2 is a plot of mean serum insulin concentrations followingi.v. administration of illustrative compositions of pegylated (5K PEGInsulin) versus non-pegylated insulin (details are provided in Example7);

[0037]FIG. 3 is a plot of blood glucose concentrations following i.v.administration of exemplary compositions of pegylated (5K PEG Insulin)versus non-pegylated insulin (details are provided in Example 7);

[0038]FIG. 4 is a plot of mean serum insulin concentrations followingintratracheal instillation of pegylated (150 μg/animal, 5K PEG Insulin)versus non-pegylated human insulin (40 μg/animal) in male rats (Example8);

[0039]FIG. 5. is a plot of mean blood glucose concentrations followingintratracheal instillation of pegylated (150 μg/animal, 5K PEG Insulin)versus non-pegylated human insulin (40 μg/animal) in male rats (Example8);

[0040]FIG. 6 is a plot of mean serum insulin concentrations followingintratracheal instillation of pegylated (750-1 PEG Insulin) versusnon-pegylated human insulin in male rats (Example 9);

[0041]FIG. 7. is a plot of mean blood glucose concentrations followingintratracheal instillation of pegylated (750-1 PEG Insulin) versusnon-pegylated human insulin in male rats (Example 9);

[0042]FIG. 8 is a plot of mean serum insulin concentrations followingintratracheal instillation of pegylated (750-1 PEG Insulin, 80 and 160μg/animal) versus non-pegylated human insulin (80 μg/animal) in malerats (Example 10);

[0043]FIG. 9. is a plot of mean blood glucose concentrations followingintratracheal instillation of pegylated (750-1 PEG Insulin, 80 and 160μg/animal) versus non-pegylated human insulin (80 μg/animal) in malerats (Example 10);

[0044]FIG. 10 is a plot of mean serum insulin concentrations followingintratracheal instillation of pegylated (750-2 PEG Insulin, 80μg/animal) versus non-pegylated human insulin (80 μg/animal) in malerats (Example 10);

[0045]FIG. 11. is a plot of mean blood glucose concentrations followingintratracheal instillation of pegylated (750-1 PEG Insulin, 80μg/animal) versus non-pegylated human insulin (80 μg/animal) in malerats (Example 11);

[0046]FIG. 12. is a plot of mean blood glucose concentrations followingintratracheal instillation of pegylated (2K PEG Insulin, 300 μg/animal,600 μg/animal, 900 μg/animal, and 1200 μg/animal) versus non-pegylatedhuman insulin (80 μg/animal) in male rats (Example 12);

[0047]FIG. 13 is a plot of mean serum insulin concentrations followingi.v. administration of an illustrative composition of pegylated (2K PEGInsulin) versus non-pegylated insulin (details are provided in Example13); and

[0048]FIG. 14 is a plot of blood glucose concentrations following i.v.administration of an exemplary composition of pegylated (2K PEG Insulin)versus non-pegylated insulin (details are provided in Example 13).

DETAILED DESCRIPTION OF THE INVENTION

[0049] The design, synthesis and characterization of variousrepresentative PEG-insulin conjugates have been optimized for pulmonarydelivery to the lung. Although the preparation of PEG-insulin moleculeshas been previously described, the use of covalent coupling of PEG forproviding prolonged action formulations of inhaleable insulin has notbeen previously demonstrated. In this regard, the challenge facing theapplicants was to provide PEG-insulin conjugates having the optimalbalance of number, location, structure, and molecular weight of PEGchains covalently attached to the insulin molecule to provide insulincompositions suitable for administration to the systemic circulation,preferably via the deep lung. Surprisingly, in light of the above, theinventors have discovered certain PEG- modified insulin formulationshaving one or more of the following features: (i) that are bioactive,i.e., that demonstrate at least about 5% of the activity of nativeinsulin, or preferably have a bioactivity that is at least eithersubstantially maintained or only minimally reduced from that of nativeinsulin, or even more preferably, having an activity that is improvedover native insulin, (ii) that are absorbed from the lung into thebloodstream (as opposed to “sticking” in the lung), (iii) that arechemically and physically stable, (iv) that, when administered to thelung, achieve blood levels of insulin that are elevated above baselinefor at least about 8 hours post administration, (v) that are resistantto enzymatic attack by insulin-degrading enzymes, and (vi) that exhibithalf lives that are extended over non-pegylated insulin whenadministered by inhalation, the details of which will become apparentwhen reading the following description.

[0050] I. Definitions

[0051] The following terms as used herein have the meanings indicated.

[0052] As used in the specification, and in the appended claims, thesingular forms “a”, “an”, “the”, include plural referents unless thecontext clearly dictates otherwise.

[0053] “Insulin” as used herein is meant to include proinsulin andencompasses any purified isolated polypeptide having part or all of theprimary structural conformation (that is to say, contiguous series ofamino acid residues) and at least one of the biological properties ofnaturally occurring insulin. In general, the term “insulin” is meant toencompass natural and synthetically-derived insulin including glycoformsthereof as well as analogs thereof including polypeptides having one ormore amino acid modifications (deletions, insertions, or substitutions)to the extent that they substantially retain at least 80% or more of thetherapeutic activity associated with full length insulin (prior tochemical modification with a hydrophilic, non-naturally occurringpolymer as described herein). The insulins of the present invention maybe produced by any standard manner including but not limited topancreatic extraction, recombinant expression and in vitro polypeptidesynthesis. Native or wild-type insulin refers to insulin having an aminoacid sequence corresponding to the amino acid sequence of insulin asfound in nature. Native or wild-type insulin can be natural (i.e.,isolated from a natural source) or synthetically produced.

[0054] A “physiologically cleavable” or “degradable” bond is a weak bondthat reacts with water (i.e., is hydrolyzed) under physiologicalconditions. Preferred are bonds that have a hydrolysis half life at pH8, 25° C. of less than about 30 minutes. The tendency of a bond tohydrolyze in water will depend not only on the general type of linkageconnecting two central atoms but also on the substituents attached tothese central atoms. Appropriate hydrolytically unstable or weaklinkages include but are not limited to carboxylate ester, phosphateester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines,orthoesters, peptides and oligonucleotides.

[0055] A “hydrolytically stable” linkage or bond refers to a chemicalbond, typically a covalent bond, that is substantially stable in water,that is to say, does not undergo hydrolysis under physiologicalconditions to any appreciable extent over an extended period of time.Examples of hydrolytically stable linkages include but are not limitedto the following: carbon-carbon bonds (e.g., in aliphatic chains),ethers, amides, urethanes, and the like. Generally, a hydrolyticallystable linkage is one that exhibits a rate of hydrolysis of less thanabout 1-2% per day under physiological conditions. Hydrolysis rates ofrepresentative chemical bonds can be found in most standard chemistrytextbooks.

[0056] “PEG” or polyethylene glycol, as used herein, is meant toencompass any water-soluble poly(alkylene oxide). Most typically, PEGsfor use in the present invention will contain the following structure,“—CH₂CH₂O(CH₂CH₂O)_(n)CH₂CH₂—, wherein the terminal groups or actualarchitecture of the overall PEG moiety may vary. One commonly employedPEG is end-capped PEG, wherein one terminus of the PEG is capped with arelatively inactive group, typically an alkoxy group such as methoxy(—OCH₃), while the other terminus is a hydroxyl group that can then besubjected to chemical modification. Specific PEG forms for use inpreparing the insulin conjugates of the invention, such as branched,linear, forked PEGs, and the like, will be described in greater detailbelow.

[0057] “PEG-insulin conjugate” refers to an insulin molecule (aspreviously defined) having covalently linked or coupled thereto at leastone polyethylene glycol moiety, and possessing any measurable degree ofinsulin activity (e.g., from about 2% to about 100% or more of thebiological activity of native insulin).

[0058] “Nominal average molecular weight” in the context of ahydrophilic, non-naturally occurring polymer of the invention such asPEG, refers to the mass average molecular weight of polymer, typicallydetermined by size exclusion chromatography, light scattering orintrinsic velocity in 1,2,4-trichlorobenzene. The polymers of theinvention are typically polydisperse, possessing a low polydispersityvalue of less than about 1.05.

[0059] A “lipophilic moiety” is one which, when attached to ahydrophilic polymer in accordance with the invention, either by adegradable or non-degradable bond, is effective to substantially alterthe hydrophilic nature of the polymer and thus the polymer-insulinconjugate. Typical lipophilic groups such as fatty acids will comprisefrom about 12-22 carbon atoms.

[0060] A “substantially non-immunogenic” insulin conjugate of theinvention possesses a reduced immunogenicity relative to native insulin.Immunogenicity may be assessed by determining antibody titres in mice orpreferably in rabbits upon administration of a PEG insulin conjugaterelative to non-modified insulin.

[0061] “Alkyl” refers to hydrocarbon chains, typically ranging about 1to 15 atoms in length. The hydrocarbon chains are preferably but notnecessarily saturated and may optionally contain additional functionalgroups attached thereto. The hydrocarbon chains may be branched orstraight chain. Exemplary alkyl groups include ethyl, propyl,1-methylbutyl, 1-ethylpropyl and 3-methylpentyl. In one preferredembodiment of the invention, conjugates comprising an alkylated PEG, andin particular, a linear alkylated PEG, are those having an alkyl portionthat is not a fatty acid or other lipophilic moiety.

[0062] “Lower alkyl” refers to an alkyl group containing from 1 to 5carbon atoms, and may be straight chain or branched, as exemplified bymethyl, ethyl, n-butyl, i-butyl, t-butyl.

[0063] “Absolute pulmonary bioavailability” is the percentage of a drugdose (e.g., of a PEG-insulin conjugate in accordance with the invention)delivered to the lungs that is absorbed and enters the blood circulationof a mammal relative to an intravenous dose of native insulin.Representative model systems for determining absolute pulmonarybioavailabilities include rat, dog, rabbit, and monkey. The inhaleablePEG-insulin compositions of the invention are, in one aspect,characterized by an absolute pulmonary bioavailability of at least about20% in plasma or blood, with absolute pulmonary bioavailabilitiesgenerally ranging from about 10% to 30% or more. Generally, dependingupon the specific nature of the PEG-insulin conjugate, a conjugate ofthe invention will possess an absolute pulmonary bioavailability of atleast about one of the following: 10%, 12%, 15%, 18%, 20%, 22%, 25%,30%, 32%, 35% or greater. Absolute pulmonary bioavailability may beestimated by measuring absorption from direct intratrachealadministration, instillation, or by inhalation of a PEG-insulinconjugate composition.

[0064] “Distribution phase”, in reference to the half-life of aPEG-insulin conjugate, refers to the initial rapid phase during whichinsulin disappears from the plasma. The terminal slow or eliminationphase half-life refers to the slow phase during which insulin iseliminated from the body.

[0065] “Prolonged effect” insulin refers to insulin having a duration ofeffect (i.e., elevated blood levels above baseline) of at least about 6hours, preferably of at least about 8 hours.

[0066] “Glucose levels that are suppressed” refers to blood levels ofglucose (e.g., after administration of a PEG-insulin conjugate of theinvention) that are suppressed below baseline or basal levels.

[0067] “Pharmaceutically acceptable salt” includes but is not limited toamino acid salts, salts prepared with inorganic acids, such as chloride,sulfate, phosphate, diphosphate, hydrobromide, and nitrate salts, orsalts prepared with an organic acid, such as malate, maleate, fumarate,tartrate, succinate, ethylsuccinate, citrate, acetate, lactate,methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate,salicylate and stearate, as well as estolate, gluceptate andlactobionate salts. Similarly salts containing pharmaceuticallyacceptable cations include, but are not limited to, sodium, potassium,calcium, magnesium, aluminum, lithium, and ammonium (includingsubstituted ammonium).

[0068] “Amino acid” refers to any compound containing both an aminogroup and a carboxylic acid group. Although the amino group mostcommonly occurs at the position adjacent to the carboxy function, theamino group may be positioned at any location within the molecule. Theamino acid may also contain additional functional groups, such as amino,thio, carboxyl, carboxamide, imidazole, etc. An amino acid may besynthetic or naturally occurring, and may be used in either its racemicor optically active (D-, or L-) forms, including various ratios ofenantiomers.

[0069] “Peptides” are composed of two or more amino acids joined by apeptide bond. Peptides can be homo- or hetero-peptides (i.e., composedof identical or different amino acid residues as defined above), and canvary in length from two amino acids to several hundred amino acids

[0070] “Dry powder” refers to a powder composition that typicallycontains less than about 10% moisture.

[0071] A composition that is “suitable for pulmonary delivery” refers toa composition that is capable of being aerosolized and inhaled by asubject so that a portion of the aerosolized particles reach the lungsto permit penetration into the lower respiratory tract and alveoli. Sucha composition is considered to be “respirable” or “inhaleable”.

[0072] “Aerosolized” particles are liquid or solid particles that aresuspended in a gas, typically as a result of actuation (or firing) of aninhalation device such as a dry powder inhaler, an atomizer, a metereddose inhaler, or a nebulizer.

[0073] “Emitted Dose” or “ED” provides an indication of the delivery ofa drug formulation from a suitable inhaler device after a firing ordispersion event. More specifically, for dry powder formulations, the EDis a measure of the percentage of powder which is drawn out of a unitdose package and which exits the mouthpiece of an inhaler device. The EDis defined as the ratio of the dose delivered by an inhaler device tothe nominal dose (i.e., the mass of powder per unit dose placed into asuitable inhaler device prior to firing). The ED is anexperimentally-determined parameter, and is typically determined usingan in-vitro device set up which mimics patient dosing. To determine anED value, a nominal dose of dry powder, typically in unit dose form, isplaced into a suitable dry powder inhaler (such as that described inU.S. Pat. No. 5,785,049, assigned to Inhale Therapeutic Systems) whichis then actuated, dispersing the powder. The resulting aerosol cloud isthen drawn by vacuum from the device, where it is captured on a taredfilter attached to the device mouthpiece. The amount of powder thatreaches the filter constitutes the emitted dose. For example, for a 5 mgdry powder-containing dosage form placed into an inhalation device, ifdispersion of the powder results in the recovery of 4 mg of powder on atared filter as described above, then the emitted dose for the drypowder composition is: 4 mg (delivered dose)/5 mg (nominaldose)×100=80%. For non-homogenous powders, ED values provide anindication of the delivery of drug from an inhaler device after firingrather than of dry powder, and are based on amount of drug rather thanon total powder weight. Similarly for MDI and nebulizer dosage forms,the ED corresponds to the percentage of drug which is drawn from adosage form and which exits the mouthpiece of an inhaler device.

[0074] “Fine particle dose” or “FPD” is defined as the mass percent ofpowder particles having an aerodynamic diameter less than 3.3 μm,typically determined by measurement in an Andersen cascade impactor.This parameter provides an indication of the percent of particles havingthe greatest potential to reach the deep lung of a patient for systemicuptake of a drug substance.

[0075] A “dispersible” or “dispersive” powder is one having an ED valueof at least about 30%, more preferably 40-50%, and even more preferablyat least about 50-60% or greater.

[0076] “Mass median diameter” or “MMD” is a measure of mean particlesize, since the powders of the invention are generally polydisperse(i.e., consist of a range of particle sizes). MMD values as reportedherein are determined by centrifugal sedimentation, although any numberof commonly employed techniques can be used for measuring mean particlesize (e.g., electron microscopy, light scattering, laser diffraction).

[0077] “Mass median aerodynamic diameter” or “MMAD” is a measure of theaerodynamic size of a dispersed particle. The aerodynamic diameter isused to describe an aerosolized powder in terms of its settlingbehavior, and is the diameter of a unit density sphere having the samesettling velocity, in air, as the particle. The aerodynamic diameterencompasses particle shape, density and physical size of a particle. Asused herein, MMAD refers to the midpoint or median of the aerodynamicparticle size distribution of an aerosolized powder determined bycascade impaction, unless otherwise indicated.

[0078] “Pharmaceutically acceptable excipient or carrier” refers to anexcipient that may optionally be included in the compositions of theinvention. Preferred for compositions for inhalation are excipients thatcan be taken into the lungs with no significant adverse toxicologicaleffects to the subject, and particularly to the lungs of the subject.

[0079] “Pharmacologically effective amount” or “physiologicallyeffective amount” is the amount of a PEG-insulin conjugate present in atherapeutic composition as described herein that is needed to provide adesired level of insulin in the bloodstream to result in a target bloodglucose level. The precise amount will depend upon numerous factors,e.g., the particular PEG-insulin, the delivery device employed, thecomponents and physical characteristics of the therapeutic composition,intended patient population, patient considerations, and the like, andcan readily be determined by one skilled in the art, based upon theinformation provided herein.

[0080] II. Hydrophilic, Non-Naturally Occurring Polymer-InsulinConjugates

[0081] Several illustrative PEG-insulin conjugates in accordance withthe invention have been prepared. Although polyethylene glycol is apreferred polymer for use in the conjugates of the invention, otherwater-soluble, hydrophilic, non-naturally occurring polymers may also beemployed. Other polymers suitable for use in the invention includepolyvinylpyrrolidone, polyvinylalcohol, polyacryloylmorpholine,polyoxazoline, and poly(oxyethylated polyols) such as poly(oxyethylatedglycerol), poly(oxyethylated sorbitol), and poly(oxyethylated glucose).Polymers comprising subunits or blocks of subunits selected from theabove-described water-soluble polymers may also be used. Additionally,Co-polymers of polyethylene glycol and polypropylene glycol may beemployed. Polymers of the invention are preferably substantially absentfatty acid groups or other lipophilic moieties.

[0082] The following section illustrates that with the careful selectionof one or more PEG moieties, pegylation reagents, insulin pegylationsites, pegylation conditions and subsequent conjugate purification,PEG-insulin compositions with the desired clinical properties (improvedpharmacokinetic and/or pharmacodynamic properties) can be obtained.Specific features of the PEG-insulin conjugates of the invention willnow be provided.

[0083] A. Structural Features of the Polymer and the Resulting Conjugate

[0084] A PEG-insulin conjugate of the invention will typically compriseone or more PEG chains each having a molecular weight ranging from about200 to about 40,000 daltons, and preferably ranging from about 200 toabout 10,000 daltons. Preferably, a PEG for use in the invention willpossess an average molecular weight falling within one of the followingranges: from about 200 to 10,000 daltons, from about 200 to about 7500daltons, from about 200 to about 6,000 daltons, from about 200 to about5,000 daltons, from about 200 to about 3000 daltons, from about 200 toabout 2000 daltons, and from about 200 to about 1000 daltons. Exemplaryconjugates prepared with PEGs having molecular weights of 5,000 daltons,2000 daltons and 750 daltons are provided in Examples 1-4.

[0085] Preferred PEG-insulins for administration to the lung willpossess a PEG moiety having a molecular weight less than about 5000daltons, preferably less than about 2000 daltons, and even less thanabout 1000 daltons. In one particular embodiment of the invention, thePEG-insulin conjugate possesses a PEG moiety having one of the followingaverage molecular weights: 200, 300, 400, 500, 600, 750, 1000, 1500,2000, 2500, 3000, 3500, 4000 or 5000. Higher molecular weight PEGs may,in certain instances, be less preferred due to a potential for loss ofactivity of the insulin molecule or, for pulmonary applications, reducedefficiency in crossing the lung (Example 8).

[0086] While lower molecular weight PEGs may be preferred for increasingbioavailability, high molecular weight PEG chains, e.g., having anaverage molecular weight of 5,000, 10,000, 15,000, 20,000, 25,000,30,000 or 40,000 daltons or greater, although generally found todecrease the bioavailability of native insulin, may be preferred forincreasing half-life, particularly in the case of injectableformulations. That is to say, a significant improvement in thepharmacokinetic parameters, e.g., the area under the curve (AUC), for ahigh molecular weight PEG insulin (relative to native), can more thancompensate for its diminished activity.

[0087] In terms of the number of subunits, PEGs for use in the inventionwill typically comprise a number of (OCH₂CH₂) subunits falling withinone or more of the following ranges: 2 to about 900 subunits, from about4 to about 200 subunits, from about 4 to about 170 subunits, from about4 to about 140 subunits, from about 4 to about 100 subunits, from about10 to about 100 subunits, from about 4 to about 70 subunits, from about4 to about 45 subunits, and from about 4 to about 25 subunits.

[0088] A PEG-insulin conjugate of the invention may be mono-substituted(i.e., that is to say, having a PEG attached to a single reactiveinsulin site) di-substituted (having PEG moieties attached to tworeactive sites, tri-substituted, or even have polymer attachments atmore than 3 sites on the insulin molecule. Mono-substituted,di-substituted, and tri-substituted insulin are also referred to hereinas PEG monomer, dimer, and trimer, respectively. Multi-substitutedinsulin (meaning insulin having PEG moieties covalently attached at 2 ormore insulin sites) will typically although not necessarily possess thesame PEG moiety attached to each reactive site. That is to say,PEG-insulin compositions having more than one type of PEG moietyattached to insulin are contemplated. Preferred compositions inaccordance with the invention are those containing predominantly monomerand/or dimer insulin conjugates. Surprisingly, PEG-insulin compositionsthat are not site-specific (comprising a mixture of PEG-insulin specieshaving PEG covalently coupled to more than one reactive site) have beenfound to possess pharmacokinetic and pharmacodynamic properties improvedover native insulin, in particular, when administered to the lung(Example 11).

[0089] With respect to the position of PEG-substitution, the insulinmolecule possesses several sites suitable for pegylation, with aminosites generally but not necessarily being most preferred. Specificinsulin amino groups suitable for pegylation include the two N-termini,GlyA1 and PheB 1, as well as LysB29. These sites on the insulin moleculeare also referred to herein simply as A1, B 1 and B29, respectively.Electrophilically activated PEGs for use in coupling to reactive aminogroups on insulin include mPEG2-ALD, mPEG-succinimidyl propionate,mPEG-succinimidyl butanoate, mPEG-CM-HBA-NHA, mPEG-benzotriazolecarbonate, mPEG-acetaldehyde diethyl acetal, and the like (ShearwaterCorporation, Huntsville, Ala.).

[0090] A composition of the invention may, in one embodiment, containpredominantly (greater than 90%) monosubstituted insulin, e.g., mono-A1insulin, mono-B1 insulin, or mono-B29 insulin. Such compositions maycontain: i) mono-A1 insulin, ii) a mixture of mono-A1 insulin andmono-B1 insulin, or iii) a mixture of mono-A1, mono-B1 and mono-B29insulin. Alternatively, a composition of the invention may containpredominantly di-substituted insulin, e.g., di-A1,B1-insulin, ordi-A1,B29-insulin, or di-B1,B29-insulin, or any of the variouscombinations thereof.

[0091] Alternatively, a composition in accordance with the invention maycontain a mixture of various PEG-insulin conjugates (i.e., PEG attachedto any one of a combination of possible attachment sites). Using theamino sites on insulin as an example, a composition of the invention maycontain any one or more of the following PEG-insulin conjugates:monoA1-PEG insulin, mono-B1-insulin, mono-B-29 insulin, di-A1,B1-insulin, di-A1,B29-insulin, di-B1,B29-insulin, ortri-A1,B-1,B29-insulin. In one embodiment, preferred are compositionscontaining predominantly monomers and dimers. Representativecompositions may comprise PEG-insulin conjugates mixtures containing atleast about 75% combined monomer and dimer, at least about 80% combinedmonomer and dimer, or at least about 85 to 90% combined monomer anddimer (e.g., Examples 5 and 6).

[0092] PheB 1 is a particularly preferred site for chemical modificationby attachment of PEG. In particular, a PEG-insulin conjugate compositionfor use in the present invention may also be characterized in oneembodiment as a composition in which at least about 70% of the B-1 siteson insulin are covalently coupled to PEG, regardless of the overallnumber of PEG-insulin species in the composition (e.g., Table 3A,Example 5). Alternative embodiments include those in which at leastabout 75% of the B-1 sites on insulin are covalently coupled to PEG, orin which at least about 80% of the B-1 sites on insulin are covalentlycoupled to PEG, or in which at least about 90% or the B-1 sites oninsulin are covalently coupled to PEG.

[0093] Surprisingly, the inventors have discovered that random mixturesof PEG-insulin (prepared by random rather than site-directedpegylation), when administered to the lung, result in elevated bloodlevels of insulin that are sustained for at least 6 hours, and moretypically for at least 8 hours or greater post-administration. Suchmixtures are advantageous not only due to their beneficialpharmacokinetic and pharmacodynamic properties, but because theirsynthesis is much simpler (does not require multiple synthetic steps,does not require the use of protecting groups, does not require multiplepurifications, etc.) than the corresponding site-specific approach.

[0094] Alternative sites in the native insulin molecule that can bechemically modified by covalent attachment of PEG include the 2C-termini, Arg22B, His10B, His5A, Glu4A, Glu17A, Glu13B, and Glu21B.

[0095] In addition to native insulin, non-native insulins having one ormore amino acid substitutions, insertions, or deletions may be utilizedsuch that additional sites become available for chemical modification byattachment of one or more PEG moieties. This embodiment of the inventionis particularly useful for introducing additional, customizedpegylation-sites within the insulin molecule, for example, for forming aPEG-insulin having improved resistance to enzymatic degradation. Such anapproach provides greater flexibility in the design of an optimizedinsulin conjugate having the desired balance of activity, stability,solubility, and pharmacological properties. Although mutations can becarried out, i.e., by site specific mutagenesis, at any number ofpositions within the insulin molecule, preferred is an insulin variantin which any one of the first four amino acids in the B-chain isreplaced with a cysteine residue. Such cysteine residues can then bereacted with an activated PEG that is specific for reaction with thiolgroups, e.g., an N-maleimidyl polymer or other derivative, as describedin U.S. Pat. No. 5,739,208 and in International Patent Publication No.WO 01/62827. Exemplary sulfhydryl-selective PEGs for use in thisparticular embodiment of the invention include mPEG-forked maleimide(mPEG(MAL)₂), mPEG2-forked maleimide (mPEG2(MAL)₂), mPEG-maleimide(mPEG-MAL), and mPEG2-maleimide (mPEG2-MAL) (Shearwater Corporation).The structures of these activated PEGS are as follows: mPEG-CONHCH[CH₂CONH(CH₂CH₂O)₂CH₂CH₂-MAL, mPEG2-lysine-NH—CH[CH₂CONH(CH₂CH₂O)₂CH₂CH₂-MAL]₂, mPEG-MAL, and mPEG2-lysine-NH—CH₂CH₂NHC(O)CH₂CH₂MAL, respectively.

[0096] Additional mutations to the native insulin sequence may beemployed, if necessary, to increase the bioactivity of a PEG-insulinconjugate whose biological activity is somewhat compromised as a resultof pegylation. One such mutation is Thr8 to a His8. Additional mutationsmay be found, for example, in Diabetes Care, 13 (9), (1990), the contentof which is herein incorporated by reference.

[0097] PEGs for use in the present invention may possess a variety ofstructures: linear, forked, branched, dumbbell, and the like. Typically,PEG is activated with a suitable activating group appropriate forcoupling a desired site or sites on the insulin molecule. An activatedPEG will possess a reactive group at a terminus for reaction withinsulin. The term “linker” as used herein is meant to encompass anactivating group positioned at a PEG terminus for reaction with insulin,and may further include additional (typically inert) atoms positionedbetween the PEG portion of the polymer and the activated group at theterminus, for ease in preparing the activated PEG. The linkers maycontain any of a number of atoms, however, preferred are linkerscontaining methylenes intervening between the PEG backbone and theterminal activating group, e.g., as in mPEG-succinimidyl propionate andmPEG-butanoate. Representative activated PEG derivatives and methods forconjugating these agents to a drug such as insulin are known in the artand further described in Zalipsky, S., et al., “Use of FunctionalizedPoly(Ethylene Glycols) for Modification of Polypeptides” in PolyethyleneGlycol Chemistry: Biotechnical and Biomedical Applications, J. M.Harris, Plenus Press, New York (1992), and in Advanced Drug Reviews,16:157-182 (1995).

[0098] In one particular embodiment of the invention, the PEG portion ofthe conjugate is absent one or more lipophilic groups effective tosignificantly modify the water-soluble nature of the polymer or of thepolymer-insulin conjugate. That is to say, the polymer or non-insulinportion of a conjugate of the invention may contain a group of atomsconsidered to be more lipophilic than hydrophilic (e.g., a carbon chainhaving from about 2 to 8 -12 carbon atoms), however, if the presence ofsuch a group or groups is not effective to significantly alter thehydrophilic nature of the polymer or of the conjugate, then such amoiety may be contained in the conjugates of the invention. That is tosay, an insulin conjugate of the invention itself is characterized ashydrophilic, rather than lipophilic or amphiphilic. Typically, thepolymer portion of an insulin conjugate, prior to coupling to insulin,whether or not containing such a lipid-loving group, will possess a highhydrophilic/lipophilic balance (HLB) number. The HLB number is basedupon a weight percentage of each type of group (hydrophilic orlipophilic) in a molecule; values typically range from about 1-40. Apolymer for use in the conjugates of the invention is, on a whole,characterized as hydrophilic, regardless of the presence of one or morelipid-loving substituents. In one embodiment of the invention, thepolymer portion of a polymer-insulin conjugate is characterized by anHLB number of greater than 25 and more preferably greater than 30, oreven more preferably greater than 35. In certain embodiments of theinvention where such a lipophilic moiety may be present, the moiety ispreferably not positioned at a terminus of a PEG chain.

[0099] Branched PEGs for use in the conjugates of the invention includethose described in International Patent Publication WO 96/21469, thecontents of which is expressly incorporated herein by reference in itsentirety. Generally, branched PEGs can be represented by the formulaR(PEG-OH)_(n), where R represents the central “core” molecule and _(n)represents the number of arms. Branched PEGs have a central core fromwhich extend 2 or more “PEG” arms. In a branched configuration, thebranched polymer core possesses a single reactive site for attachment toinsulin. Branched PEGs for use in the present invention will typicallycomprise fewer than 4 PEG arms, and more preferably, will comprise fewerthan 3 PEG arms. Branched PEGs offer the advantage of having a singlereactive site, coupled with a larger, more dense polymer cloud thantheir linear PEG counterparts. One particular type of branched PEG canbe represented as (MeO-PEG-)_(p)R—X, where p equals 2 or 3, R is acentral core structure such as lysine or glycerol having 2 or 3 PEG armsattached thereto, and X represents any suitable functional group that isor that can be activated for coupling to insulin. One particularlypreferred branched PEG is mPEG2-NHS (Shearwater Corporation, Alabama)having the structure mPEG2-lysine-succinimide.

[0100] In yet another branched architecture, “pendant PEG” has reactivegroups for protein coupling positioned along the PEG backbone ratherthan at the end of PEG chains as in the previous example. The reactivegroups extending from the PEG backbone for coupling to insulin may bethe same or different. Pendant PEG structures may be useful but aregenerally less preferred, particularly for compositions for inhalation.

[0101] Alternatively, the PEG-portion of a PEG-insulin conjugate maypossess a forked structure having a branched moiety at one end of thepolymer chain and two free reactive groups (or any multiple of 2) linkedto the branched moiety for attachment to insulin. Exemplary forked PEGsare described in International Patent Publication No. WO 99/45964, thecontent of which is expressly incorporated herein by reference. Theforked polyethylene glycol may optionally include an alkyl or “R” groupat the opposing end of the polymer chain. More specifically, a forkedPEG-insulin conjugate in accordance with the invention has the formula:R-PEG-L(Y-insulin)_(n), where R is alkyl, L is a hydrolytically stablebranch point and Y is a linking group that provides chemical linkage ofthe forked polymer to insulin, and n is a multiple of 2. L may representa single “core” group, such as “—CH—”, or may comprise a longer chain ofatoms. Exemplary L groups include lysine, glycerol, pentaerythritol, orsorbitol. Typically, the particular branch atom within the branchingmoiety is carbon.

[0102] In one particular embodiment of the invention, the linkage of theforked PEG to the insulin molecule, (Y), is hydrolytically stable. In apreferred embodiment, n is 2. Suitable Y moieties, prior to conjugationwith a reactive site on insulin, include but are not limited to activeesters, active carbonates, aldehydes, isocyanates, isothiocyanates,epoxides, alcohols, maleimides, vinylsulfones, hydrazides,dithiopyridines, and iodacetamides. Selection of a suitable activatinggroup will depend upon the intended site of attachment on the insulinmolecule and can be readily determined by one of skill in the art. Thecorresponding Y group in the resulting PEG-insulin conjugate is thatwhich results from reaction of the activated forked polymer with asuitable reactive site on insulin. The specific identity of such thefinal linkage will be apparent to one skilled in the art. For example,if the reactive forked PEG contains an activated ester, such as asuccinimide or maleimide ester, conjugation via an amine site on insulinwill result in formation of the corresponding amide linkage. Theseparticular forked polymers are particularly attractive since theyprovide conjugates having a molar ratio of insulin to PEG of 2:1 orgreater. Such conjugates may be less likely to block the insulinreceptor site, while still providing the flexibility in design toprotect the insulin against enzymatic degradation, e.g., by insulindegrading enzyme.

[0103] In a related embodiment, a forked PEG-insulin conjugate of theinvention is represented by the formula: R-[PEG-L(Y-insulin)₂]_(n). Inthis instance R represents a central core structure having attachedthereto at least one PEG-di-insulin conjugate. Specifically, preferredforked polymers in accordance with this aspect of the invention arethose were n is selected from the group consisting of 1,2,3,4,5,and 6.Exemplary core R structures may also be derived from lysine, glycerol,pentaerythritol, or sorbitol.

[0104] In an alternative embodiment, in any of the representativestructures provided herein, the chemical linkage between insulin and thepolymer branch point may be degradable (i.e., hydrolytically unstable).Alternatively, one or more degradable linkages may be contained in thepolymer backbone to allow generation in vivo of a PEG-insulin conjugatehaving a smaller PEG chain than in the initially administered conjugate.Such optional features of the polymer conjugate may provide foradditional control over the final desired pharmacological properties ofthe conjugate upon administration. For example, a large and relativelyinert conjugate (i.e., having one or more high molecular weight PEGchains attached thereto, e.g., one or more PEG chains having a molecularweight greater than about 10,000, wherein the conjugate possessesessentially no bioactivity) may be administered, which then either inthe lung or in the bloodstream, is hydrolyzed to generate a bioactiveconjugate possessing a portion of the originally present PEG chain. Inthis way, the properties of the PEG-insulin conjugate may be somewhatmore effectively tailored. For instance, absorption of the initialpolymer conjugate may be slow upon initial administration, which ispreferably but not necessarily by inhalation. Upon in-vivo cleavage ofthe hydrolytically degradable linkage, either free insulin (dependingupon the position of the degradable linkage) or insulin having a smallpolyethylene tag attached thereto, is then released and more readilyabsorbed through the lung and/or circulated in the blood.

[0105] In one feature of this embodiment of the invention, the intactpolymer-conjugate, prior to hydrolysis, is minimally degraded uponadministration, such that hydrolysis of the cleavable bond is effectiveto govern the slow rate of release of active insulin into thebloodstream, as opposed to enzymatic degradation of insulin prior to itsrelease into the systemic circulation.

[0106] Appropriate physiologically cleavable linkages include but arenot limited to ester, carbonate ester, carbamate, sulfate, phosphate,acyloxyalkyl ether, acetal, and ketal. Such conjugates should possess aphysiologically cleavable bond that is stable upon storage and uponadministration. For instance, a PEG-cleavable linkage-insulin conjugateshould maintain its integrity upon manufacturing of the finalpharmaceutical composition, upon dissolution in an appropriate deliveryvehicle, if employed, and upon administration irrespective of route.

[0107] More particularly, as described generally above, PEG-insulinconjugates having biodegradable linkages and useful in the presentinvention are represented by the following structures:PEG1-W-PEG2-insulin (where PEG1 and PEG2 can be the same or different)or PEG-W-insulin wherein W represents a weak, biodegradable linkage.These conjugates contain PEG arms or portions of PEG arms that areremovable (i.e., cleavable) in-vivo. These particular modified insulinsare typically substantially biologically inactive when intact, eitherdue to the size of the intact PEG-portion of the molecule or due tosteric blockage of the active sites on the insulin molecule by the PEGchain. However, such conjugates are cleaved under physiologicalconditions to thereby release insulin or a biologically activePEG-insulin capable of absorption into the systemic circulation, e.g.,from the lung. In a first exemplary structure, the PEG1 portion maypossess any of a number of different architectures discussed herein, andwill typically possess a molecular weight of at least about 10,000, suchthat the conjugate is not rapidly absorbed upon administration. The PEG2portion of the molecule preferably possesses a molecular weight of lessthan about 5000 daltons, more preferably less than 2000 daltons, andeven more preferably less than 1000 daltons. Referring now to thesecondary exemplary structure, PEG-W-insulin, the PEG portion willgenerally possess a molecular weight of at least about 10,000 Daltons ormore.

[0108] In yet another specific embodiment of the invention, thePEG-insulin conjugate has a dumbbell-like structure in which two insulinmoieties are interconnected by a central PEG. More specifically, suchconjugates may be represented by the structure insulin-Y-PEG-Z-insulin,where Y and Z are hyrolytically stable linking groups linking insulin tothe PEG moiety. In a particular embodiment, Z is an activated sulfone,which prior to conjugation, is suitable for reaction with thiol groupson insulin (e.g., cysteines). Alternatively, Y and Z may be any groupsuitable for covalent coupling with insulin. Additional examples areprovided in U.S. Pat. No. 5,900,461, the content of which is expresslyincorporated herein by reference.

[0109] Additional representative mono-and di-functional PEGs havingeither linear or branched structures for use in preparing the conjugatesof the invention may be purchased from Shearwater Corporation (Alabama).Illustrative structures are described in Shearwater's 2001 catalogueentitled “Polyethylene Glycol and Derivatives for BiomedicalApplications”, the contents of which is expressly incorporated herein byreference.

[0110] B. Preparation

[0111] The reaction conditions for coupling PEG to insulin will varydepending upon the particular PEG derivative employed, the site ofattachment on insulin and the particular type of reactive group (i.e.,lysine versus cysteine), the desired degree of pegylation, and the like,and can readily be determined by one skilled in the art.

[0112] As exemplified in greater detail below, synthesis of theconjugates of the invention may be site-directed (Examples 1, 2 and 4)or may be random (Example 3). Suitable PEG activating groups forreaction with insulin amine groups (e.g., GlyA1, PheB1, Lys29B), aretresylate, aldehyde, epoxide, carbonylimidazole, active carbonates (e.g.succinimidyl carbonate), acetal, and active esters such asN-hydroxylsuccinimide (NHS) and NHS-derivatized PEGs . Of these, themost reactive are PEG carboxymethyl-NHS, norleucine-NHS, andsuccinimidyl carbonate. Additional PEG reagents for coupling to insulininclude PEG succinimidyl succinate and propionate. PEG active esterssuitable for use in the invention, e.g., having a single propanoic orbutanoic acid moiety, are described in U.S. Pat. No. 5,672,662, thecontents of which is incorporated herein in its entirety. Specificactive esters for use in preparing the conjugates of the inventioninclude mPEG-succinimidyl propionate and mPEG-succinimidyl butanoate(Examples 1-4).

[0113] Optimized experimental conditions for a specific conjugate canreadily be determined, typically by routine experimentation, by oneskilled in the art.

[0114] Reactive groups suitable for activating a PEG-polymer forattachment to a thiol (sulfhydryl) group on insulin includevinylsulfones, iodoacetamide, maleimide, and dithio-orthopyridine.Particularly preferred reagents include PEG vinylsulfones andPEG-maleimide. Additional representative vinylsulfones for use in thepresent invention are described in U.S. Pat. No. 5,739,208, the contentof which is expressly incorporated herein by reference.

[0115] In some instances, the compositions of the invention compriseselectively PEGylated insulin, i.e., the resulting conjugates areessentially homogeneous with respect to the position and degree ofpegylation. That is to say, site selective or site directed pegylationof an amino group will result in an insulin conjugate compositionwherein primarily the intended target position, e.g., PheB1, has a PEGmoiety attached thereto. Depending upon the intended site of pegylation,a protection/deprotection synthetic strategy may be necessary to preventpegylation of non-target reactive sites within the insulin molecule,e.g., by employing a protecting group such as t-BOC(tert-butoxycarbonyl) or di-BOC (di- butoxycarbonyl). Other suitableamino protecting groups include carbobenzoxy (CBZ), trityl derivativessuch as trityl (Tr), dimethoxytrityl (DMTr) and the like. Otherprotecting groups, such as cyclic diacyl groups or nitrophenylsulfenyl(Nps) may also prove useful for protecting amino functions. An exemplarysite-directed synthesis of a 5K-PEG-insulin composition is provided inExamples 1 and 2.

[0116] Such site directed coupling chemistry employed to provide theinsulin conjugates of the invention results in compositions having alarge degree of substitution at a particular reactive site on theinsulin molecule. These compositions can then, if desired, be furtherpurified to provide compositions of essentially pure mono- ordi-functional PEG-insulins.

[0117] An essentially pure PEG-insulin composition refers to onecomprising a PEG-insulin conjugate that is at least about 90% pure, andpreferably at least about 95% pure by any one of the followinganalytical methods. In this respect, purity refers to PEG-insulinconjugate content. That is to say, a PEG-insulin conjugate that is atleast about 90% pure contains at least about 90% by weight ofPEG-insulin conjugate species, while the other nearly 10% representsimpurities that are not PEG-insulin conjugate. PEG-insulin conjugates ofthe invention are typically purified using one or more purificationtechniques such as ion exchange chromatography, size exclusionchromatography, affinity chromatography, hydrophobic interactionchromatography, and reverse phase chromatography. The overallhomogeneity of the resulting PEG-insulin (number of insulin-PEG formspresent) can be assessed using one or more of the following methods:chromatography, electrophoresis, mass spectrometry, and in particular,MALDI-MS, and NMR spectroscopy. One particularly useful method foridentifying the sites of insulin modification is RP-HPLC peptidemapping, coupled with a USP identity test for human insulin usingendoproteinase Glu-C (Example 6).

[0118] C. Characteristics of PEG-insulin Conjugates

[0119] In accordance with one aspect of the invention, provided arePEG-insulin conjugate compositions that are suitable for pulmonaryadministration. As can be seen by the in-vivo data in Examples 7-11, thePEG insulin conjugates of the invention, when administered to the lung,possess pharmacokinetic and pharmodynamic properties improved overnative insulin. It has been shown that insulin can be modified with PEGshaving a molecular weight of up to 5,000K to 10,000 K or greater, andstill maintain activity. Activity of a representative PEG-insulinconjugate, 5K-PEG-insulin, is demonstrated in Example 7. Additionally,as can be seen from the examples provided herein, exemplary PEG-insulinconjugates possessing PEG chains with average molecular weights rangingfrom 750 daltons, to 2,000 daltons, to 5,000 daltons, when administeredboth intravenously and to the lung, are bioactive, are not substantiallyheld up within the lung when administered to the lung, as evidenced bydetectable serum levels of insulin, and are effective in producing asubstantial suppression of glucose (Examples 7 to 11), which, in certaincases, is over a duration of time significantly greater than thatobserved for native insulin. Moreover, provided herein are PEG-insulinconjugates, which when administered to the lung, exhibit a rapid onsetof action (within 1 hour of administration). A summary ofpharmacokinetic and pharmacodynamic parameters for exemplary PEG-insulincompositions of the invention is provided in Table 13.

[0120] In general, a PEG-insulin composition of the invention willpossess one or more of the following characteristics. The PEG-insulinconjugates of the invention maintain at least a measurable degree ofspecific activity. That is to say, a PEG-insulin conjugate in accordancewith the invention will possesses anywhere from about 2% to about 100%or more of the specific activity of native insulin. In one preferredembodiment of the invention, the PEG-insulin conjugate will possess atleast 10% or more of the biological activity of unmodified, nativeinsulin and is substantially non-immunogenic. Preferably, thebioactivity of a conjugate of the invention will range from about 5% toat least about 20% or more of the bioactivity of native insulin. Thebioactivity of a conjugate of the invention may be characterizedindirectly, e.g., by monitoring blood glucose and insulin levels togenerate the corresponding pharmacodynamic and/or pharmacokinetic data,or by RIA (radioimmunoassay).

[0121] In considering serum concentrations of insulin followingadministration of a PEG-insulin conjugate, e.g., to the lung, theconjugates described herein will typically peak (i.e., reach Cmax or thehighest point in the concentration curve) at from around 2 to 8 hourspost dose, and more typically will peak at around 3 to 6 hours or so.Moreover, the chemically modified insulins of the invention, and inparticular, the prolonged effect insulin formulations provided herein,are effective in providing both a measurable glucose-lowering effect andsustained concentrations of insulin over a longer period of time thannative insulin. More specifically, a PEG-insulin conjugate whenadministered to the lung will exhibit elevated levels of insulin(elevated over basal or baseline levels) for at least about 6 hours andpreferably for at least 8 hours post administration. Preferably, aPEG-insulin conjugate when administered to the lung, results in elevatedblood levels of insulin over a prolonged period of at least 9 hours, 10hours, 12 hours or at least 14 hours post administration whereinabove-basal levels of insulin conjugate are detectable in thebloodstream for such an extended duration post dose. Representativecompositions demonstrating these features are provided in the Examples.

[0122] As described previously, an insulin conjugate of the invention iseffective to lower blood glucose levels. Turning now to the ability ofthe compositions of the invention to suppress blood glucose, a PEGinsulin conjugate when administered, e.g., to the lung, is effective tosuppress blood glucose levels below basal levels for at least 6 hourspost-administration. More particularly, a PEG-insulin composition of theinvention is effective to suppress blood glucose levels below baselinefor at least 8 hours, preferably for at least 10 hours, or morepreferably for at least 12 hours or more post administration.

[0123] Moreover, the PEG-insulin formulations of the invention exhibitabsolute pulmonary bioavailabilities that are improved over nativeinsulin. Specifically, a PEG-insulin formulation as provided hereinpossesses an absolute pulmonary bioavailability that is at least about1.2 times that of native insulin, preferably at least about 1.5 timesthat of native insulin, more preferably is at least about 2 timesgreater or even more preferably is at least about 2.5 or 3 times greaterthan that of native insulin. (Illustrative results are provided in Table13).

[0124] III. Formulations

[0125] The polymer-insulin conjugate compositions of the invention maybe administered neat or in therapeutic/pharmaceutical compositionscontaining additional excipients, solvents, stabilizers, etc., dependingupon the particular mode of admistration and dosage form. The presentconjugates may be administered parenterally as well as non-parenterally.Specific administration routes include oral, rectal, buccal, topical,nasal, ophthalmic, subcutaneous, intramuscular, intraveneous,transdermal, and pulmonary. Most preferred are parenteral and pulmonaryroutes.

[0126] Pharmaceutical formulations for mammalian and preferably humanadministration will typically comprise at least one PEG-insulinconjugate of the invention together with one or more pharmaceuticallyacceptable carriers, as will be described in greater detail below,particularly for pulmonary compositions. Formulations of the presentinvention, e.g., for parenteral administration, are most typicallyliquid solutions or suspensions, while inhaleable formulations forpulmonary administration are generally liquids or powders, with powderformulations being generally preferred. Additional albeit less preferredcompositions of the chemically modified insulins of the inventioninclude syrups, creams, ointments, tablets, and the like.

[0127] Formulations and corresponding doses of insulin will vary withthe concentration bioactivity of the insulin employed. Injectableinsulin is measured in USP Insulin Units and USP Insulin Human Units(U); one unit of insulin is equal to the amount required to reduce theconcentration of blood glucose in a fasting rabbit to 45 mg/dl (2.5 mM).Typical concentrations of insulin preparations for injection range from30-100 Units/mL which is about 3.6 mg of insulin per mL. The amount ofinsulin required to achieve the desired physiological effect in apatient will vary not only with the particulars of the patient and hisdisease (e.g., type I vs. type II diabetes) but also with the strengthand particular type of insulin used. For instance, dosage ranges forregular insulin (rapid acting) are from about 2 to 0.3 U insulin perkilogram of body weight per day. Compositions of the invention are, inone aspect, effective to achieve in patients undergoing therapy afasting blood glucose concentration between about 90 and 140 mg/dl and apostprandial value below about 250 mg/dl. Precise dosages can bedetermined by one skilled in the art when coupled with thepharmacodynamics and pharmacokinetics of the precise insulin-conjugateemployed for a particular route of administration, and can readily beadjusted in response to periodic glucose monitoring.

[0128] Individual dosages (on a per inhalation basis) for inhaleableinsulin-conjugate formulations are typically in the range of from about0.5 mg to 15 mg insulin-conjugate, where the desired overall dosage istypically achieved in from about 1-10 breaths, and preferably in fromabout 1 to 4 breaths. On average, the overall dose of PEG-insulinadministered by inhalation per dosing session will range from about 10Uto about 400U, with each individual dosage or unit dosage form(corresponding to a single inhalation) containing from about 5U to 400U.

[0129] A. Inhaleable Formulations of Chemically Modified Insulin

[0130] As stated above, one preferred route of administration for theinsulin conjugates of the invention is by inhalation to the lung.Particular formulation components, characteristics and delivery deviceswill now be more fully described.

[0131] The amount of insulin conjugate in the formulation will be thatamount necessary to deliver a therapeutically effective amount ofinsulin per unit dose to achieve at least one of the therapeutic effectsof native insulin, i.e., the ability to control blood glucose levels tonear normoglycemia. In practice, this will vary widely depending uponthe particular insulin conjugate, its activity, the severity of thediabetic condition to be treated, the patient population, the stabilityof the formulation, and the like. The composition will generally containanywhere from about 1% by weight to about 99% by weight PEG-insulin,typically from about 2% to about 95% by weight conjugate, and moretypically from about 5% to 85% by weight conjugate, and will also dependupon the relative amounts of excipients/additives contained in thecomposition. More specifically, the composition will typically containat least about one of the following percentages of PEG-insulin: 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more by weight. Preferably,powder compositions will contain at least about 60%, e.g., from about60-100% by weight PEG-insulin. It is to be understood that more than oneinsulin may be incorporated into the formulations described herein andthat the use of the term “agent” or “insulin” in no way excludes the useof two or more insulins or a combination of insulin with another activeagent. (For example, an illustrative PEG-insulin formulation may alsocomprise native insulin).

[0132] A.1. Excipients

[0133] Compositions of the invention will, in most instances, includeone or more excipients. Preferred are carbohydrate excipients, eitheralone or in combination with other excipients or additives.Representative carbohydrates for use in the compositions of theinvention include sugars, derivatized sugars such as alditols, aldonicacids, esterified sugars, and sugar polymers. Exemplary carbohydrateexcipients suitable for use in the invention include, for example,monosaccharides such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitolsorbitol (glucitol), pyranosyl sorbitol, myoinositol and the like.Preferred are non-reducing sugars, sugars that can form a substantiallydry amorphous or glassy phase when combined with an insulin conjugate,and sugars possessing relatively high Tgs (e.g., Tgs greater than 40°C., preferably greater than 50° C., more preferably greater than 60° C.,and even more preferably greater than 70° C., and most preferably havingTgs of 80° C. and above).

[0134] Additional excipients include amino acids, peptides andparticularly oligomers comprising 2-9 amino acids, and more preferably2-5 mers, and polypeptides, all of which may be homo or hetero species.Representative amino acids include glycine (gly), alanine (ala), valine(val), leucine (leu), isoleucine (ile), methionine (met), proline (pro),phenylalanine (phe), trytophan (trp), serine (ser), threonine (thr),cysteine (cys), tyrosine (tyr), asparagine (asp), glutamic acid (glu),lysine (lys), arginine (arg), histidine (his), norleucine (nor), andmodified forms thereof. One particularly preferred amino acid isleucine.

[0135] Also preferred for use as excipients in inhaleable compositionsare di- and tripeptides containing two or more leucyl residues, asdescribed in Inhale Therapeutic System's International patentapplication PCT/US00/09785, incorporated herein by reference in itsentirety.

[0136] Also preferred are di- and tripeptides having a glass transitiontemperature greater than about 40° C., more preferably greater than 50°C., even more preferably greater than 60° C., and most preferablygreater than 70° C.

[0137] Although less preferred due to their limited solubility in water,additional stability and aerosol performance-enhancing peptides for usein the invention are 4-mers and 5-mers containing any combination ofamino acids as described above. More preferably, the 4-mer or 5-mer willcomprise two or more leucine residues. The leucine residues may occupyany position within the peptide, while the remaining (i.e., non-leucyl)amino acids positions are occupied by any amino acid as described above,provided that the resulting 4-mer or 5-mer has a solubility in water ofat least about 1 mg/ml. Preferably, the non-leucyl amino acids in a4-mer or 5-mer are hydrophilic amino acids such as lysine, to therebyincrease the solubility of the peptide in water.

[0138] Polyamino acids, and in particular, those comprising any of theherein described amino acids, are also suitable for use as stabilizers.Preferred are polyamino acids such as poly-lysine, poly-glutamic acid,and poly(lys, ala).

[0139] Additional excipients and additives useful in the presentcompositions and methods include but are not limited to proteins,non-biological polymers, and biological polymers, which may be presentsingly or in combination. Suitable excipients are those provided inInhale Therapeutic Systems' International Publication Nos. WO 96/32096and 98/16205. Preferred are excipients having glass transitiontemperatures (Tg), above about 35° C., preferably above about 40° C.,more preferably above 45° C., most preferably above about 55° C.

[0140] Exemplary protein excipients include albumins such as human serumalbumin (HSA), recombinant human albumin (rHA), gelatin, casein,hemoglobin, and the like. The compositions may also include a buffer ora pH adjusting agent, typically but not necessarily a salt prepared froman organic acid or base. Representative buffers include organic acidsalts of citric acid, ascorbic acid, gluconic acid, carbonic acid,tartaric acid, succinic acid, acetic acid, or phthalic acid. Othersuitable buffers include Tris, tromethamine hydrochloride, borate,glycerol phosphate and phosphate. Amino acids such as glycine are alsosuitable.

[0141] The compositions of the invention may also include additionalpolymeric excipients/additives, e.g., polyvinylpyrrolidones, derivatizedcelluloses such as hydroxymethylcellulose, hydroxyethylcellulose, andhydroxypropylmethylcellulose, Ficolls (a polymeric sugar),hydroxyethylstarch (HES), dextrates (e.g., cyclodextrins, such as2-hydroxypropyl-β-cyclodextrin and sulfobutylether-β-cyclodextrin),polyethylene glycols, and pectin.

[0142] The compositions may further include flavoring agents,taste-masking agents, inorganic salts (e.g., sodium chloride),antimicrobial agents (e.g., benzalkonium chloride), sweeteners,antioxidants, antistatic agents, surfactants (e.g., polysorbates such as“TWEEN 20” and “TWEEN 80”, and pluronics such as F68 and F88, availablefrom BASF), sorbitan esters, lipids (e.g., phospholipids such aslecithin and other phosphatidylcholines, phosphatidylethanolamines,although preferably not in liposomal form), fatty acids and fattyesters, steroids (e.g., cholesterol), and chelating agents (e.g., EDTA,zinc and other such suitable cations). The use of certain di-substitutedphosphatidylcholines for producing perforated microstructures (i.e.,hollow, porous microspheres) is described in greater detail below. Otherpharmaceutical excipients and/or additives suitable for use in thecompositions according to the invention are listed in “Remington: TheScience & Practice of Pharmacy”, 19^(th) ed., Williams & Williams,(1995), and in the “Physician's Desk Reference”, 52^(nd) ed., MedicalEconomics, Montvale, N.J. (1998).

[0143] In one embodiment, a composition in accordance with the inventionmay be absent penetration enhancers, which can cause irritation and aretoxic at the high levels often necessary to provide substantialenhancement of absorption. Specific enhancers, which may be absent fromthe compositions of the invention, are the detergent-like enhancers suchas deoxycholate, laureth-9, DDPC, glycocholate, and the fusidates.Certain enhancers, however, such as those that protect insulin fromenzyme degradation, e.g., protease and peptidase inhibitors such asalpha-i antiprotease, captropril, thiorphan, and the HIV proteaseinhibitors, may, in certain embodiments of the invention, beincorporated in the PEG-insulin formulations of the invention. In yetanother embodiment, the PEG-insulin conjugates of the invention may beabsent liposomes, lipid matrices, and encapsulating agents.

[0144] Generally, the pharmaceutical compositions of the invention willcontain from about 1% to about 99% by weight excipient, preferably fromabout 5%-98% by weight excipient, more preferably from about 15-95% byweight excipient. Even more preferably, the spray dried composition willcontain from about 0-50% by weight excipient, more preferably from 0-40%by weight excipient. In general, a high insulin concentration is desiredin the final pharmaceutical composition. Typically, the optimal amountof excipient/additive is determined experimentally, i.e., by preparingcompositions containing varying amounts of excipients (ranging from lowto high), examining the chemical and physical stability of thePEG-insulin, MMADs and dispersibilities of the pharmaceuticalcompositions, and then further exploring the range at which optimalaerosol performance is attained with no significant adverse effect uponinsulin stability.

[0145] A.2. Preparing Dry Powders

[0146] Dry powder formulations of the invention comprising a PEG-insulinconjugate may be prepared by any of a number of drying techniques, andpreferably by spray drying. Spray drying of the formulations is carriedout, for example, as described generally in the “Spray Drying Handbook”,5^(th) ed., K. Masters, John Wiley & Sons, Inc., NY, N.Y. (1991), and inPlatz, R., et al., International Patent Publication Nos. WO 97/41833(1997) and WO 96/32149 (1996), the contents of which are incorporatedherein by reference.

[0147] Solutions of PEG-insulin conjugates are spray dried in aconventional spray drier, such as those available from commercialsuppliers such as Niro A/S (Denmark), Buchi (Switzerland) and the like,resulting in a dispersible, dry powder. Optimal conditions for spraydrying the PEG-insulin solutions will vary depending upon theformulation components, and are generally determined experimentally. Thegas used to spray dry the material is typically air, although inertgases such as nitrogen or argon are also suitable. Moreover, thetemperature of both the inlet and outlet of the gas used to dry thesprayed material is such that it does not cause degradation of thePEG-insulin in the sprayed material. Such temperatures are typicallydetermined experimentally, although generally, the inlet temperaturewill range from about 50° C. to about 200° C., while the outlettemperature will range from about 30° C. to about 150° C. Preferredparameters include atomization pressures ranging from about 20-150 psi,and preferably from about 30-40 to 100 psi. Typically the atomizationpressure employed will be one of the following (psi): 20, 30, 40, 50,60, 70, 80, 90, 100, 110, 120 or above.

[0148] Respirable PEG-insulin compositions having the features describedherein may also be produced by drying certain formulation componentswhich result in formation of a perforated microstructure powder asdescribed in WO 99/16419, the entire contents of which are incorporatedby reference herein. The perforated microstructure powders typicallycomprise spray-dried, hollow microspheres having a relatively thinporous wall defining a large internal void. The perforatedmicrostructure powders may be dispersed in a selected suspension media(such as a non-aqueous and/or fluorinated blowing agent) to providestabilized dispersions prior to drying. The use of relatively lowdensity perforated (or porous) microstructures or microparticulatessignificantly reduces attractive forces between the particles, therebylowering the shear forces, increasing the flowability and dispersibilityof the resulting powders, and reducing the degradation by flocculation,sedimentation or creaming of the stabilized dispersions thereof.

[0149] Alternatively, a PEG-insulin composition for pulmonary deliverymay comprise aerodynamically light particles as described in U.S. Pat.No. 6,136,295.

[0150] A powdered formulation of the invention may also be prepared bylyophilization, vacuum drying, spray freeze drying, super critical fluidprocessing (e.g., as described in Hanna, et al., U.S. Pat. No.6,063,138), air drying, or other forms of evaporative drying.

[0151] In yet another approach, dry powders may be prepared byagglomerating the powder components, sieving the materials to obtainagglomerates, spheronizing to provide a more spherical agglomerate, andsizing to obtain a uniformly-sized product, as described, e.g., and inAhlneck, C., et al., International PCT Publication No. WO 95/09616,1995, incorporated herein by reference.

[0152] Dry powders may also be prepared by blending, grinding, sievingor jet milling formulation components in dry powder form.

[0153] Once formed, the dry powder compositions are preferablymaintained under dry (i.e., relatively low humidity) conditions duringmanufacture, processing, and storage. Irrespective of the drying processemployed, the process will preferably result in inhaleable, highlydispersible particles comprising a chemically modified insulin asdescribed herein.

[0154] A.3. Features of Dry Powder Formulations

[0155] Powders of the invention are further characterized by severalfeatures, most notably, one or more of the following: (i) consistentlyhigh dispersibilities, which are maintained, even upon storage (ii)small aerodynamic particles sizes (MMADs), (iii) improved fine particledose values, i.e., powders having a higher percentage of particles sizedless than 3.3 microns MMAD, all of which contribute to the improvedability of the powder to penetrate to the tissues of the lowerrespiratory tract (i.e., the alveoli) for delivery to the systemiccirculation. These physical characteristics of the inhaleable powders ofthe invention, to be described more fully below, are important inmaximizing the efficiency of aerosolized delivery of such powders to thedeep lung.

[0156] Dry powders of the invention are composed of aerosolizableparticles effective to penetrate into the lungs. The particles of theinvention have a mass median diameter (MMD) of less than about 20-30 μm,or less than 20 μm, or less than about 10 μm, preferably less than about7.5 μm, and more preferably less than about 4 μm, and even less thanabout 3.5 μm, and usually are in the range of 0.1 μm to 5 μm indiameter. Preferred powders are composed of particles having an MMD fromabout 0.2 to 4.0 μm. In some cases, the powder will also containnon-respirable carrier particles such as lactose, where thenon-respirable particles are typically greater than about 40 microns insize.

[0157] The powders of the invention are further characterized by anaerosol particle size distribution less than about 10 μm mass medianaerodynamic diameter (MMAD), preferably having MMADs less than about 5μm, more preferably less than 4.0 μm, even more preferably less than 3.5μm, and most preferably less than 3 μm. The mass median aerodynamicdiameters of the powders will characteristically range from about 0.1-10μm, preferably from about 0.2-5.0 μm MMAD, more preferably from about1.0-4.0 μm MMAD, and even more preferably from about 1.5 to 3.0 μm.Small aerodynamic diameters can generally be achieved by a combinationof optimized spray drying conditions and choice and concentration ofexcipients.

[0158] The PEG-insulin powders of the invention may further becharacterized by their densities. A powdered composition for inhalationwill generally possess a bulk density from about 0.1 to 10 g/cubiccentimeter, preferably from about 0.1-2 g/cubic centimeter, and morepreferably from about 0.15-1.5 g/cubic centimeter.

[0159] The powders will generally have a moisture content below about20% by weight, usually below about 10% by weight, and preferably belowabout 5% by weight. Preferred powders in accordance with the inventionhaving a moisture content that is below about one or more of thefollowing weight percentages: 15%, 10%, 7%, 5%, or 3%. Such lowmoisture-containing solids tend to exhibit a greater stability uponpackaging and storage.

[0160] Additionally, the spray drying methods and stabilizers describedherein are effective to provide highly dispersible PEG-insulinformulations. For powder formulations, the emitted dose (ED) of thesepowders is typically greater than 30%, and usually greater than 40%.More preferably, the ED of the powders of the invention is greater than50%, and is often greater than 60%.

[0161] The compositions described herein also possess good stabilitywith respect to both chemical stability and physical stability, i.e.,aerosol performance over time. Generally, with respect to chemicalstability, the PEG-insulin conjugate contained in the formulation willdegrade by no more than about 15% upon spray drying. That is to say, thepowder will possess at least about 85% intact PEG-insulin conjugate,preferably at least about 90 or 95% intact conjugate, and even morepreferably will contain at least about 97% or greater intactPEG-insulin. Preferably, the spray drying process will result in powdershaving less than about 10% total protein aggregates, that is to say,greater than 90% by weight of the chemically modified insulin being inmonomeric form.

[0162] With respect to aerosol performance, compositions of theinvention are generally characterized by a drop in emitted dose of nomore than about 20%, preferably no more than about 15%, and morepreferably by no more than about 10%, when stored under ambientconditions for a period of three months.

[0163] A.4. Administration of the Composition

[0164] PEG-insulin formulations as described herein may be deliveredusing any suitable dry powder inhaler (DPI), i.e., an inhaler devicethat utilizes the patient's inhaled breath as a vehicle to transport thedry powder drug to the lungs. Preferred are Inhale Therapeutic Systems'dry powder inhalation devices as described in Patton, J. S., et al.,U.S. Pat. No. 5,458,135, Oct. 17, 1995; Smith, A. E., et al., U.S. Pat.No. 5,740,794, Apr. 21, 1998; and in Smith, A. E., et. al., U.S. Pat.No. 5,785,049, Jul. 28, 1998, herein incorporated by reference. Whenadministered using a device of this type, the powdered medicament iscontained in a receptacle having a puncturable lid or other accesssurface, preferably a blister package or cartridge, where the receptaclemay contain a single dosage unit or multiple dosage units. Convenientmethods for filling large numbers of cavities (i.e., unit dose packages)with metered doses of dry powder medicament are described, e.g., inParks, D. J., et al., International Patent Publication WO 97/41031, Nov.6, 1997, incorporated herein by reference.

[0165] Other dry powder dispersion devices for pulmonary administrationof dry powders include those described, for example, in Newell, R. E.,et al, European Patent No. EP 129985, Sep. 7, 1988; in Hodson, P. D., etal., European Patent No. EP472598, Jul. 3, 1996; in Cocozza, S., et al.,European Patent No. EP 467172, Apr. 6, 1994, and in Lloyd, L. J. et al.,U.S. Pat. No. 5,522,385, Jun. 4, 1996, incorporated herein by reference.Also suitable for delivering PEG-insulin dry powders are inhalationdevices such as the Astra-Draco “TURBUHALER”. This type of device isdescribed in detail in Virtanen, R., U.S. Pat. No. 4,668,218, May 26,1987; in Wetterlin, K., et al., U.S. Pat. No. 4,667,668, May 26, 1987;and in Wetterlin, K., et al., U.S. Pat. No. 4,805,811, Feb. 21, 1989,all of which are incorporated herein by reference. Other suitabledevices include dry powder inhalers such as Rotahaler® (Glaxo), Discus®(Glaxo), Spiros™ inhaler (Dura Pharmaceuticals), and the Spinhaler®(Fisons). Also suitable are devices which employ the use of a piston toprovide air for either entraining powdered medicament, liftingmedicament from a carrier screen by passing air through the screen, ormixing air with powder medicament in a mixing chamber with subsequentintroduction of the powder to the patient through the mouthpiece of thedevice, such as described in Mulhauser, P., et al, U.S. Pat. No.5,388,572, Sept. 30, 1997, incorporated herein by reference.

[0166] An inhaleable PEG-insulin composition may also be delivered usinga pressurized, metered dose inhaler (MDI), e.g., the Ventolin® metereddose inhaler, containing a solution or suspension of drug in apharmaceutically inert liquid propellant, e.g., a chlorofluorocarbon orfluorocarbon, as described in Laube, et al., U.S. Pat. No. 5,320,094,Jun. 14, 1994, and in Rubsamen, R. M., et al, U.S. Pat. No. 5,672,581(1994), both incorporated herein by reference.

[0167] Alternatively, the PEG-insulins described herein may be dissolvedor suspended in a solvent, e.g., water or saline, and administered bynebulization. Nebulizers for delivering an aerosolized solution includethe AERx™ (Aradigm), the Ultravent® (Mallinkrodt), the Pari LC Plus™ orthe Pari LC Star™ (Pari GmbH, Germany), the DeVilbiss Pulmo-Aide, andthe Acorn II (Marquest Medical Products).

[0168] As previously described, the PEG-insulin conjugates describedherein can also be administered parenterally by intravenous injection,or less preferably by intramuscular or by subcutaneous injection.Precise components of such formulations can be readily determined by oneskilled in the art. Suitable formulation types for parenteraladministration include ready-for-injection solutions, dry powders forcombination with a solvent prior to use, suspensions ready forinjection, dry insoluble compositions for combination with a vehicleprior to use, emulsions and liquid concentrates for dilution prior toadministration. For instance, an injectable solution of a PEG-insulincomposition of the invention may include the composition dissolved in anaqueous vehicle such as aqueous sodium chloride, Ringers solution, adextrose-injection solution, lactated Ringers solution and the like, andmay include one or more pharmaceutically acceptable compatibleexcipients or additives as described above.

[0169] IV. Utility

[0170] The compositions of the invention are useful, when administeredby any suitable route of administration, and preferably by inhalation orby injection, in a therapeutically effective amount to a mammaliansubject, for treating diabetes mellitus, and in particular, type I ortype II diabetes.

[0171] All articles, books, patents and other publications referencedherein are hereby incorporated by reference in their entirety.

[0172] The following examples illustrate, but in no way are intended tolimit the scope of the present invention.

EXAMPLES

[0173] Materials and Methods

[0174] Polyethylene glycol reagents were obtained from ShearwaterCorporation (Huntsville, Ala.).

[0175] Human insulin was obtained from Diosynth, Inc.

Example 1 Synthesis of Di-N^(αA1),N^(εB29)-t-Boc-Insulin

[0176] A composition composed primarily of mono-pegylated insulin wasprepared in a site-specific fashion as set forth in Examples 1 and 2using an exemplary linear 5,000 Dalton polyethylene glycol.

[0177] Di-protected insulin was first prepared as follows. 602 mg ofhuman insulin (0.103 mmol) was dissolved in 3.0 mL of anhydrous dimethylsulfoxide (DMSO) containing 166 uL of triethylamine. 50□1 ofdi-tert-butyldicarbonate (0.215 mmol) was added to the insulin solution.After 60 min at room temperature, the reaction solution was poured into240 mL of acetone followed by addition of 3 drops of 6 M HCl to initiateflocculation. The precipitate was isolated by filtration, and dried invacuo. The reaction product was purified by preparative HPLC using aWaters 25×100 mm C18 column (mean particle size, 15 μm; pore size,100A). Mixtures of acetonitrile and 0.1%TFA in deionized water were usedas eluents at a rate of 3.0 mL/min. The product was collected, distilledto remove acetonitrile, and then lyophilized. Yield was 164.8 mg (26.7%,MW ˜6000 by MALDI).

Example 2 Synthesis of Mono-Pegylated mPEG-5K-SPA-PheB1-InsulinConjugate N^(αBI)-Methoxypoly(ethylene glycol)5K-insulin(mPEG5K-PheB1-insulin)

[0178] 150 mg (˜0.025 mmol) of newly purified di-N^(αA1),N^(εB29)-t-boc-Insulin from Example 1 was dissolved in 4 mL of DMSOcontaining 95 μL of triethylamine. 169 mg (0.032 mmol) of mPEG-SPA-5000(mPEG-succinimidyl propionate, mPEG-O-CH ₂CH₂C(O)O-succinimide, MW5,000) was added to the insulin solution. After incubation overnight (29hours) at room temperature, the resulting mPEG-insulin derivative wasdiluted to 100 mL with D.I. water, dialyzed against D.I. water for 4hours and then lyophilized. The lyophilized product was re-dissolved in4 mL of anhydrous TFA and maintained under N₂ at 0° C. for 1.5 hours toremove the Boc protecting groups. The deprotected mPEG-insulin wasdiluted to 50 mL with D.I. water and dialyzed against 0.1% NH₄HCO₃ andD.I. water overnight. Lyophilization of the product yielded a whitepowder. Yield was 117.6 mg (41.6%, MW˜11311.6 by MALDI).

[0179] The percentage of mono-conjugated insulin, based upon massspectral data, was approximately 90%, confirming the site specificnature of this synthetic approach. Additional characterization data isprovided in Example 5. Insulin content of the resulting product was51.3%. For ease of reference, N^(αB1)-Methoxypoly(ethylene glycol)5K-propionamido-insulin or mPEG5K-PheB1-insulin will be referred to hereinas “5K PEG insulin”.

Example 3 Synthesis of mPEG-2K-SPA-Insulin Conjugate

[0180] The following approach was utilized to prepare insulin pegylatedin a non-site specific (i.e., random) fashion utilizing an exemplarylinear polyethylene glycol having a molecular weight of approximately2,000 Daltons. 0.1012 g of insulin (MW 5826 Da, 0.01737 mmol) wasdissolved in 0.5 mL of anhydrous DMSO and treated with 50 μL oftriethylamine (0.3587 mmol, 20 fold molar excess). To the above reactionmixture was added 52 mg of m-SPA-2000 (mPEG-succinimidyl propionate,Shearwater Corporation, MW˜2000 Da, 0.02605 mmol, 1.5 fold molarexcess). The mixture was stirred for about 17 hours at room temperatureunder nitrogen. The reaction mixture was then dissolved in 0.1% TFA to atotal volume of 5.5 mL and purified by reverse phase IHPLC using a C-18column, and acetonitrile/0.1%TFA as eluent). Reverse phase HPLC revealeda mixture of both mono (one PEG attached) and di-pegylated (two PEGsattached) product; the composition is referred to herein as “2K PEGinsulin”.

[0181] Yield: 68 mg

[0182] Insulin content by RP-HPLC: 50.5 mg

Example 4 Synthesis of mPEG-750 Da -SPA-Insulin Conjugate

[0183] A composition composed predominantly of insulin pegylated at theB1 site was prepared in a site-specific fashion using a representativepolyethylene glycol modifier, i.e, a linear 750 Dalton polyethyleneglycol having a succinimidyl propionate terminus suitable for covalentattachment to insulin.

[0184] 4A. Synthesis of Di-N^(A1),N^(B29)-t-Boc-Insulin

[0185] Di-protected insulin was prepared as follows. 602 mg of humaninsulin (0.103 mmol) was dissolved in 3.0 mL of anhydrous dimethylsulfoxide (DMSO) containing 166 uL of triethylamine. 50ul ofdi-tert-butyldicarbonate (0.215 mmol) was added to the insulin solution.After 60 min at room temperature, the reaction solution was poured into240 mL of acetone followed by addition of 3 drops of 6 M HCl to initiateflocculation. The precipitate was isolated by filtration and dried invacuo. The reaction product was purified by preparative HPLC using aWaters 25×100 mm C18 column (mean particle size, 15um; pore size, 100A).Mixtures of acetonitrile and 0.1%TFA in deionized water were used aseluents at a rate of 3.0 mL/min. The product was collected, distilled toremove acetonitrile, and then lyophilized. Yield was 164.8 mg (26.7%,MW˜6000 by MALDI).

[0186] 4B. Synthesis of mPEG-750 Da-SPA-PheB1-Insulin Conjugate

[0187] 63.4 mg (˜0.01056 mmol) of newly purifieddi-N^(A1),N^(B29)-t-boc-Insulin from Example 4A was dissolved in 0.5 mLof DMSO containing 200 uL of triethylamine. 33 mg (0.03173 mmol, MW ofmSPA750 is about 1040Da) of mPEG-SPA-750 (mPEG-succinimidyl propionate,mPEG—O—CH2CH2C(O)O-succinimide, PEG MW 750) was added to the insulinsolution. After incubation overnight (29 hours) at room temperature, 300uL of TFA was added to the reaction mixture and the resultingmPEG-insulin derivative was precipitated in 100 mL ethyl ether and driedunder vacuum. Yield was about 28.5 mg with 21.3 mg of insulin contentmeasured by reverse phase HPLC (33.6%, MW˜6639.3Da by MALDI. Forsimplicity, the composition is referred to herein as “750 PEG Insulin”.

[0188] Two different syntheses were carried out on this material bothutilizing the above synthetic methodology with one exception: onesynthesis was carried out at a molar ratio of mPEG-SPA-750 to insulin of7:1 while the other synthesis was conducted at a molar ratio ofmPEG-SPA-750 to insulin of 3:1. The product compositions resulting fromthese two preparations are referred to herein as “750-1 PEG insulin”(molar ratio of PEG reagent to insulin was 7:1) and “750-2 PEG insulin”(molar ratio of PEG reagent to insulin was 3:1).

Example 5 Characterization of Exemplary PEG-Insulin Compositions

[0189] The pegylated insulin conjugate compositions described above werefurther characterized by various analytical techniques.

[0190] Mass spectrometry was utilized to provide an estimate of therelative amounts of mono, di, and tri-conjugated insulin (also referredto as PEG insulin monomer, dimer, and trimer) present in each of thecompositions based upon relative peak areas. The results are provided inTable 1 below. TABLE 1 Relative Amounts of Mono, Di, and Tri-ConjugatedInsulin Based on Mass Spectrometry PEG-Insulin Composition %Monoconjugate % Diconjugate % Triconjugate 5K PEG insulin 91  4 notdetermined 750-1 PEG 46 39 15 insulin 750-2 PEG 60 32  8 insulin 2K PEGinsulin 51 45 not determined

[0191] Size exclusion chromatography (SEC) was carried out on the 750-1,750-2 and 2K PEG-insulin compositions described above employing twoShodex SEC columns (part number KW-802.5) assembled in series on aWaters 2690 HPLC system. The mobile phase consisted of 22% glacialacetic acid and 33% acetonitrile (V/V) in water. The chromatography datawas used as an alternative approach for determining the relative amountsof mono, di, and tri-conjugated insulin in each of these compositions.The results are presented in Table 2 below. As can be seen by acomparison of the data in Tables 1 and 2, the two different methodsprovide results that are in close agreement with respect to the relativeamounts of each type of conjugate present in the compositions. TABLE 2Relative Amounts of Mono, Di, and Tri-Conjugated Insulin Based on HP-SECInsulin % Type % Monoconjugate % Diconjugate % Triconjugate Other PEG750-1 48 47 5 0 PEG 750-2 66 26 7 2 PEG 2000 40 51 nd 9

[0192] Additional studies were carried out to determine the distributionof the various positional conjugates in each of three exemplarycompositions, i.e., the extent of substitution at each of the threepossible attachment sites, A-1Gly, B-1Phe or B-29 Lys. Dithiothreitol(DTT, Sigma) was used to reduce the disulfide bonds in the insulinsamples, causing the covalent attachments between the insulin A and Bchains to be broken.

[0193] To carry out the reduction reactions, the PEG-insulin sampleswere dissolved in 8 M urea containing 0.4 M ammonium bicarbonate atabout 0.2 mg/mL of equivalent insulin mass for each conjugated species.DTT was dissolved in water (7 mg/ml) to form an aqueous DTT solution.One part of DTT solution was then added to 5 parts of each of theinsulin solutions, and the reduction reaction was carried out at 50° C.for 15 minutes. The reduced 750 PEG insulin compositions were alkylatedwith iodoacetamide (Sigma). Six parts of the PEG-insulin solutions werereacted with 1 part of 100 mM iodoacetamide prior to chromatography andenzyme digestion. The reaction products were then analyzed by HPLC.Percent conjugation to either the A or B chain of insulin was estimatedbased upon the amount of insulin A or B chain that eluted later than thecontrol (and attributed to conjugation to polyethylene glycol). Theselate-eluting peaks were therefore missing at the expected retentiontimes for the control. Relative peak areas were used to provide anindication of the percent conjugation of polyethylene glycol to eitherthe A or B chain of insulin.

[0194] To further explore the relative amount of PEG attached to B-29Lys versus B 1-Phe, the reduced and alkylated A and B chains of the750-1 and 750-2 PEG insulin compositions from the DTT-reductionsdescribed above were further digested with the sequencing grade enzymeEndoproteinase Glu-C (Sigma). A solution containing the enzyme at 0.125μg/μL in aqueous ammonium bicarbonate was prepared. Prior to enzymesolution addition, the insulin concentration in each of the reducedreaction mixtures was 0.05 μg/μL in 8 M urea containing 0.4 M ammoniumbicarbonate. 1 part enzyme solution was then added to 40 parts insulinsolution. Digestion with Endoproteinase Glu-C enzyme produces theinsulin peptide fragments of A1-A4, A5-A17, A18-A21, B1-B13, B14-B21,AND B22-B30.

[0195] Fragments resulting from the enzymatic digests for both the A andB chains of the 750-1 and 750-2 PEG insulin compositions were analyzedby HPLC to estimate the overall distribution of PEG attachment sites toinsulin for each of these compositions. The percent of the peak missingrelative to the control provided an estimation of the amount of thefragment conjugated to PEG, since that fragment eluted elsewhere on thechromatogram. TABLE 3A Distribution of PEG Attachment Sites forExemplary PEG-Insulin Formulations % of A1 Sites % of B-1 sites % ofB-29 Sites Insulin type Conjugated Conjugated Conjugated PEG 750-1 30 9521 PEG 750-2 11 95 15 PEG 2000 63 85*

[0196] The numbers in Table 3A are based on the possibility of each sitebeing 100 percent conjugated. For example, each mono-species has 3possible configurations (mono-A1, mono-B1, mono-B-29) and eachdiconjugate has 3 configurations (di-A1, B-1; di-A-1,B-29; and di-B-1,B-29). Looking at the data in Table 3A, e.g., for PEG-750-1, over all ofthe possible species present in the composition, 95% of the PEG-insulinconjugates possess a polyethylene glycol covalently attached at the B-1site. TABLE 3B Various Conjugate Species Possible TRI- TypesMONO-conjugate DI-conjugate conjugate HMWP Species # 1 2 3 4 5 6 7 8Point of A-1 B-1 B-29 (A-1 + (A-1 + (B-1 + (A-1 + Conjugated ConjugationB-1) B-29) B-29) B-1 + Insulin B-29) Dimers

Example 6 Comparison of the Rate of Enzymatic Digestion of 750-2 PEGInsulin Versus Unmodified Insulin

[0197] The rate of enzymatic digestion of 750-2 PEG-insulin bychymotrypsin was compared to that of insulin.

[0198] An insulin control and a PEG 750 insulin-2 composition wasprepared at 1 mg/mL in phosphate buffered saline solution at pH 7.8.Chymostrypsin was prepared at 1 mg/mL in 1 mM HCl solution. 1 part ofthe enzyme solution was added to 20 parts of the insulin solution. Smallaliquots of the mixed solution were withdrawn approximately every hour.

[0199] A RP(reverse phase)-HPLC method was developed using a C-18 columnwith a mobile phase containing sodium perchlorate, phosphoric acid, andacetonitrile. An acetonitrile gradient was employed to elute theassortment of PEGylated insulin species in a group of slightly resolvedpeaks monitored at 214 nm. The group of peaks were integrated manuallyas one peak and labeled as PEG insulin. As the digestion proceeded, theloss of intact PEG insulin and insulin were plotted (FIG. 1). Theirhalf-life in the presence of chymotrypsin was estimated.

[0200] The length of time required for enzymatic digestion of half ofthe concentration of the main component of the 750-2 PEG insulincomposition was five times greater than for unmodified insulin. That isto say, it took five times longer for digestion by chymostrypsin of halfthe concentration of the illustrative pegylated insulin than for regularinsulin. These results demonstrate the potential of PEG insulinconjugates for prolonged residence time in the alveoli due to enhancedresistance to proteolytic degradation when compared to unmodifiedinsulin.

Example 7 Evaluation of Serum Glucose and Insulin ConcentrationsFollowing Intraveneous Administration of 5K PEG Insulin in Rats(P-2001-015)

[0201] This study was conducted to determine whether the activity ofinsulin in the 5K PEG insulin composition was retained upon chemicalmodification with an exemplary 5K polyethylene glycol chain, and toexplore the dose and glucose response curves for these compositions whenadministered intraveneously.

[0202] Pre-cannulated (jugular/femoral vein [JVC/FVC]) male SpragueDawley Rats (325-350 grams) having an access port threaded under theskin externalizing at the nape of the neck were supplied by Hilltop LabAnimals Inc. (P.O. Box 183, Scottdale, Pa. 15683). The jugular cannulaswere filled with a solution (lumen filler) of pharmaceutical gradePolyviny-Pyrrolidone (PVP—MW 40,000), physiological saline and sodiumheparin to retain patency. The nylon filament plug sealing the cannulawas removed and replaced with a Monoject blunt cannula 23G×1 (VWR#53498-484) on the day of the study. The test system included 1 male ratrandomly chosen for the placebo group, 2 male rats randomly chosen forthe non-pegylated group, and 4 male rats randomly chosen for thePEG-insulin group. The source of pegylated insulin for this study was 5KPEG insulin from Example 2. Doses were administered intraveneously.

[0203] Number/Sex of Animals

[0204] Day 1: IM/Group for the placebo Group 1

[0205]2M/Group for Group 2; 4M/Group for Groups 3-5

[0206] The animals were fasted for 12-18 hours prior to the initiationof the study. Human insulin (Diosynth) was stored at −20° C. prior touse. 5K PEG Insulin (Example 2) was stored at −20° C. prior to use. Twodifferent solutions for administration were prepared:

[0207] Solutions for I.V. Administration

[0208] Non-PEGylated Human Insulin (1.0 mg/ml Stock): 1.0 ml of PBS wasadded to 1.0 mg of insulin powder.

[0209] 5K PEG Insulin: (1.0 mg/ml Human Insulin-concentration is basedupon insulin rather than on the conjugate): 6.0 ml of PBS was added to11.7 mg of 5K PEG Insulin powder.

[0210] The animals were anesthetized with inhaled isoflurane. The i.v.doses (300 μL/animal) were given through the FVC and then the catheterwas tied off to eliminate cross contamination with the blood draws. Allblood samples were drawn through the JVC. Phosphate buffered saline(PBS) was administered to Group 1 as a 300 μL i.v. dose. Non-pegylatedhuman insulin was administered to Group 2 as a 20 μg/animal i.v. dose.The PEGylated human insulin formulation was administered to Group 3 as a20 μg/animal i.v. dose, Group 4 as a 40 μg/animal i.v. dose, and Group 5as a 30 μg/animal i.v. dose. Blood samples (˜500 μL) were collected fromthe JVC at predose (2 to 0.25 hours prior to dosing), 10, 15, 30, 60,120, and 180 minutes postdose. A small amount of blood was placed on aglucose test strip for determination of blood glucose by the GlucometerElite glucose monitor (Bayer Corp., Elkart, Ind.). The remainder of thesample was placed into serum separator tubes and placed into thecentrifuge to separate the blood. The serum was then decanted into aseparate tube and analyzed by radioimmunoassay (RIA). Means and standarddeviations (SD) were calculated using Microsoft® Excel 2000. TABLE 4Summary of In-Vivo Experiments The following are the actual dosesadministered and actual animal numbers per group that were used. Thestudy was completed in one day. Total Daily Dose of Number of GroupRoute of Insulin Animals/ Number Composition Administration (□g/animal)Gender 1 Placebo i.v.  0 1M 2 Non-PEG Insulin i.v. 20 2M 3 PEG Insulini.v. 20 4M 4 PEG Insulin i.v. 40 4M 5 PEG Insulin i.v. 30 4M

[0211] The results demonstrate that the 5K PEG insulin compositionpossesses bioactivity, i.e., the insulin molecule remains active uponmodification with polyethylene glycol, as can be seen by its ability tolower blood glucose. Mean serum insulin concentrations following i.v.administration of pegylated insulin were dose dependent; a dosedependent decrease in glucose levels was also observed. The results aresummarized in FIGS. 2 and 3. FIG. 2 is a plot of mean serum insulinconcentrations following i.v. administration of illustrativecompositions of pegylated versus non-pegylated insulin; FIG. 3 is a plotof blood glucose concentrations following i.v. administration of thecompositions described above.

Example 8 Administration of 5K PEG Insulin to the Lung (P-2001-017)

[0212] An exemplary pegylated insulin, 5K PEG insulin, was administeredto rats via intratracheal administration to determine (i) whether itsactivity was maintained upon administration to the lung, and (ii) itsimpact, if any, on serum insulin and blood glucose concentrations whendelivered directly to the lung.

[0213] Stock Solutions

[0214] Non-PEGylated Insulin: 1 ml of PBS was added to 1.0 mg of insulinpowder to prepare a 1 mg/ml stock solution. The stock solution ofinsulin (control) was prepared on the study initiation day.

[0215] 5K PEG Insulin: 4.0 ml of PBS was added to 7.8 mg of 5K PEGInsulin powder to prepare a 1 mg/ml (based on insulin) stock solution.

[0216] Dosing Solutions

[0217] 40 μg/animal of Insulin: Within 2 hours of dosing, 667 μl of theinsulin stock solution was added to 4.33 ml of PBS.

[0218] 150 μg/animal of Insulin B-1: Within 2 hours of dosing, 2.5 ml ofthe 5K PEG Insulin stock solution was added to 2.5 ml of PBS.

[0219] Intratracheal Instillation

[0220] The rats were lightly anesthetized using inhaled 3.0-5.0%Isoflurane (Abbott Laboratories) mixed with oxygen for approximately 5minutes in a plexiglass anesthesia chamber. Administration wasaccomplished by insertion of a gavage needle (Popper & Sons Inc.; 18×3″W2-¼ mm ball, New Hyde Park, N.Y. 11040) fitted into a 1 mL syringe intothe mouth of the rat down the trachea to just above the main carina.When inserting the gavage needle into the trachea, proper insertion wasdetected by feeling for the roughness of the cartilage rings under theskin of the throat using the ball of the gavage needle. Doses wereadministered into the lungs utilizing this method, and followed byremoval of the gavage needle.

[0221] Fourteen (N=7/Group) fasted male rats (Hilltop Lab Animals,Scottsdale, Pa. (300-350 g)) with indwelling jugular vein catheters(JVC) were used for this study. Non-pegylated human insulin wasadministered to Group 1 as a 40□g/300 □L i.t. dose. The PEGylated humaninsulin formulation was administered to Group 2 as a 150 □g/300 □L i.t.dose. Blood samples (˜500 □L) were collected at predose (2 to 0.25 hoursprior to dosing), 15, 30, 60, 120, 240, 360, 480, and 720 minutespostdose. A small amount of blood was placed on a glucose test strip fordetermination of blood glucose in the Glucometer Elite glucose monitor(Bayer Corp., Elkart, Ind.). The remainder of the sample was placed intoserum separator tubes and analyzed by radioimmunoassay. Means andstandard deviations (SD) were calculated using Microsoft□ Excel 2000.Animal 2-3 was dropped from the study due to a clogged catheter. TABLE 5Summary of In-Vivo Experiments in Rats The following are the actualdoses administered and actual animal numbers per group that were used.Total Route of Number of Daily Dose No of Type of Adminis- Animals/ ofInsulin Dosing Group No. Insulin tration Gender (μg/animal) Days 1Insulin I.T. 7M  40 1 2 5K PEG I.T. 7M 150 1 Insulin

[0222] TABLE 6 In-Vivo Dose Levels Total Daily Dose Dose Concentrationof Group Type of of Insulin Volume Dosing No. Insulin (μg/animal) (μl)Solution (μg/ml) 1 Insulin  40 300 133.33 2 5K PEG 150 300 500 Insulin

[0223] Mean serum concentrations and mean blood concentrations ofinsulin and 5K PEG Insulin following intratracheal administration wereplotted and are shown in FIG. 4 and in FIG. 5 respectively. The resultsdemonstrate that the pegylated insulin compositions of the inventionpossess activity upon delivery to and residence within the lung. Thepharmacokinetic data further demonstrate that pegylated insulin not onlypasses through the lung into the circulation, but does so whilstmaintaining activity, as evidenced by detectable serum insulin levelscorresponding to non-endogenous insulin. Due to the blood levels ofinsulin observed within about 1 hour following intratrachealadministration, it appears that pegylated insulin is not substantiallyheld up within the lungs and crosses the lungs into the bloodstreamshortly after administration. The results further indicate thatpegylated insulin, when administered to the lung, is effective inlowering blood glucose. However, in the present example, pegylatedinsulin appears less effective than non-pegylated insulin at the dosesadministered in lowering blood glucose. Both the pharmacokinetic andpharmacodynamic response curves for intratracheally administeredpegylated insulin somewhat resemble non-pegylated insulin, althoughbased upon the profiles in FIG. 4, PEG-insulin appears to be longeracting than non-pegylated insulin. Further optimization of the dosingamounts and particular polyethylene glycol modifiers may be readilyachieved by one of skill in the art, based upon the guidance presentedherein and depending upon the dosing requirements, intended patientpopulation, condition to be treated, and the like, of a particularchemically modified insulin product.

Example 9 Administration of 750-1 PEG Insulin to the Lung (P-2001-025)

[0224] A representative pegylated insulin, 750-1-PEG Insulin, wasadministered to rats by intratracheal administration. The study wasconducted, in part, to explore the effects of a composition of insulincovalently attached to one or more polyethylene glycol chains having anapproximate molecular weight of 1,000 Daltons or less, when administeredto the lung.

[0225] The in-vivo rat study was conducted essentially as described inExample 8 above. The precise dosing regimen followed and dosesadministered are summarized in the tables below. TABLE 7 Total Route ofNumber of Daily Dose No of Type of Adminis- Animals/ of Insulin DosingGroup No. Insulin tration Gender (μg/animal) Days 1 Insulin I.T. 2M  801 2 750-1 I.T. 4M 100 1 PEG Insulin 3 750-1 I.T. 4M 300 1 PEG Insulin 4750-1 I.T. 4M 500 1 PEG Insulin

[0226] TABLE 8 Total Daily Dose Dose Concentration of Group Type of ofInsulin Volume Dosing No. Insulin (μg/animal) (μl) Solution (μg/ml) 1Insulin  80 300 266.67 2 750-1 100 300 333.33 PEG Insulin 3 750-1 300300 1000.00 PEG Insulin 4 750-1 500 300 1666.67 PEG Insulin

[0227] Serum insulin and blood glucose concentrations of insulin and750-1 PEG Insulin following intratracheal administration in rats wereplotted and are shown in FIG. 6 and FIG.7 respectively. When looking atthe plot of mean serum insulin concentrations in FIG. 6, native ornon-pegylated insulin reached its maximum serum concentration atapproximately 15 minutes, while the pegylated insulin compositionsreached maximum serum concentrations at 6 hours (100 μg/animal) and 8hours (300 μg/animal), demonstrating the long-acting nature of thesecompositions when administered to the lung by inhalation. As can be seenin FIG. 6, unmodified insulin returned to baseline at approximately 6hours post administration while the insulin levels for pegylated insulinat 6 hours were significantly above baseline (from about 3 to 7 times orgreater the baseline value). Moreover, intratracheal administration ofpegylated insulin resulted in sustained levels of systemic insulin thathad not returned to baseline even at 12 hours post administration. Infact, insulin levels for pegylated insulin were over three times thebaseline value (i.e., the value for non-modified insulin) at both 8 and12 hours. A plot of these results is shown in FIG. 6.

[0228] In sum, when administered to the lung, 750-1 PEG insulin resultedin increased systemic insulin levels when compared to unmodifiedinsulin. Moreover, systemic insulin levels for the pegylated insulingroup were still significantly above baseline even at 12 hours. That isto say, elevated insulin levels were sustained for the pegylated insulingroup for at least twice as long as for unmodified insulin. This datafurther demonstrates that pegylated insulin crosses the lungs, isbioactive, and provides prolonged systemic levels of insulin whencompared to unmodified insulin.

[0229] A plot of mean blood glucose concentrations followingintratracheal administration of non-pegylated versus 750-1 pegylatedinsulin is provided in FIG. 7. Blood glucose response levels correlatednicely with serum insulin levels for the pegylated insulin group. (Thatis to say, at elevated levels of serum insulin, a correspondingsupression/lowering of blood glucose was also observed). In looking atFIG. 7, it can be seen that the pegylated insulin compositions of theinvention, when administered orally to the lung, exhibit a rapid onsetof action comparable to native insulin rather than a delayed onset ofaction typical of many sustained release formulations. That is to say,suppression of glucose occurs shortly after administration.Additionally, while native or non-pegylated insulin reaches maximumglucose lowering in about 2 hours, the time to reach maximum glucoselowering for pegylated insulin was extended to at least 4 hours, 6 hoursand 8 hours for the 500 μg, 100 μg, and 300 μg doses, respectively. So,the time to reach maximum blood glucose lowering for pegylated insulin,when administered to the lung, was prolonged 2 to 4 times over that ofnon-pegylated insulin. Overall, the 750-1 PEG insulin glucosesuppression was significantly increased over the 12 hour period whencompared with unmodified insulin. At 8 hours, glucose levels hadessentially returned to normal for unmodified insulin, while glucoselevels for the PEG insulin group were from 1.3 to 3 times lower than forunmodified insulin. Glucose levels for the pegylated insulin group hadnot returned to even at 12 hours, further indicating prolonged glucosesuppression for the lly derivatized insulin compositions of the presentinvention.

Example 10 Administration of 750-1 PEG Insulin to the Lung (P-2002-001)

[0230] In a study similar to Example 9 above, 750-1-PEG insulin wasadministered to intratracheal administration at doses lower than thoseemployed in Example 9.

[0231] The in-vivo intratracheal rat study was conducted essentially asdescribed in Example 8 above. The precise dosing regimen and dosesadministered are summarized in the tables below. TABLE 9 Total Route ofNumber of Daily Dose No of Type of Adminis- Animals/ of Insulin DosingGroup No. Insulin tration Gender (μg/animal) Days 1 Insulin i.t. 5M 80 12 750-1 i.t. 5M 80 1 PEG Insulin 3 750-1 i.t 5M 160 1 PEG Insulin

[0232] TABLE 10 Total Daily Dose Dose Concentration of Group Type of ofInsulin Volume Dosing No. Insulin (μg/animal) (μl) Solution (μg/ml) 1Insulin  80 300 266.7 2 750PEG  80 300 266.7 Insulin-1 3 750PEG 160 300533.3 Insulin-1

[0233] Serum insulin and blood glucose concentrations of unmodifiedinsulin and 750-1 PEG insulin following intratracheal administration inrats were plotted and the results are shown in FIG. 8 and in FIG. 9,respectively. When looking at the plot of mean serum insulinconcentrations in FIG. 8, native or non-pegylated insulin reached itsmaximum serum concentration at approximately 15 minutes, while thepegylated insulin compositions reached maximum serum concentrations at 2hours (80 μg/animal) and 6 hours (160 μg/animal). That is to say, thetime to reach maximum serum levels of insulin for peg-modified insulinwas extended 8 to 24 times over native or non-pegylated insulin whenadministered to the systemic circulation via the lung. As can be seen inFIG. 8, unmodified insulin returned to baseline at approximately 12hours post administration, while insulin levels for the PEG-insulingroup ranged from 2.5 to 3.5 times the baseline value at the same 12hour time point. Insulin levels for the pegylated insulin group did notreturn to baseline until around 25 hours, meaning that it took twice aslong for the pegylated insulin group to return to baseline when comparedto unmodified insulin. Systemic insulin levels were sustained for thepegylated insulin group for a duration of time about two-fold or twice(25 hours versus 12 hours) that of unmodified insulin. At time points upto about 6 hours, the insulin levels for the two pegylated insulingroups roughly corresponded to the doses administered (that is to say,insulin concentrations for the 160 μg/animal group were approximatelytwice those of the 80 μg/animal group).

[0234] A plot of mean blood glucose concentrations followingintratracheal administration of non-pegylated versus 750-1 pegylatedinsulin is provided in FIG. 9. At 25 hours post administration, glucosesuppression for both pegylated insulin groups had still not returned tobaseline in contrast to unmodified insulin. Similar to the results fromExample 9, the overall profiles for pegylated insulin demonstrateprolonged glucose suppression extending beyond 25 hours. At 8 hours,glucose levels had returned to nearly normal for unmodified insulinwhile glucose levels for the PEG insulin groups were about 1.5 timeslower than for unmodified insulin. These results further demonstratethat modifying insulin with one or more polyethylene glycol moietiesresults in good bioavilability across the lungs and prolonged systemicinsulin levels as well as prolonged glucose suppression.

Example 11 Administration of 750-2 PEG Insulin to the Lung (P-2002-003)

[0235] A representative pegylated insulin composition, 750-2-PEGinsulin, was administered to rats by intratracheal administration. Thisstudy was conducted to further explore the effect of various doses ofpegylated versus non-pegylated insulin when administered directly to thelungs. Animals were dosed at 80 μg insulin per animal for both pegylatedand non-pegylated forms of insulin. The in-vivo rat study was conductedessentially as described in Example 8 above. The precise dosing regimenfollowed and doses administered are summarized in the tables below.TABLE 11 Total Route of Number of Daily Dose No of Type of Adminis-Animals/ of Insulin Dosing Group No. Insulin tration Gender (μg/animal)Days 1 Insulin IT 7M 80 1 2 750PEG-2 IT 7M 80 1 Insulin

[0236] TABLE 12 Total Daily Dose Dose Concentration of Group Type of ofInsulin Volume Dosing No. Insulin (μg/animal) (μl) Solution (μg/ml) 1Insulin 80 300 266.7 2 750PEG-2 80 300 266.7 Insulin

[0237] A plot of mean serum insulin concentrations followingintratracheal instillation of both non-pegylated and 750-1 PEG insulinat a dose of 80 μg/animal is shown in FIG. 10. A plot of mean bloodglucose concentrations following intratracheal instillation of bothnon-pegylated and 750-1 PEG insulin at a dose of 80 μg/animal is shownin FIG. 11. Results similar to those in Examples 9 and 10 were obtained.

[0238] A tabulation of pharmacokinetic parameters from Examples 10 and11 is provided below. Bioavailability is absolute bioavailability (i.e.,compared to intraveneously administered insulin). TABLE 13 Mean SerumInsulin Pharmacokinetics AUC μU * Dose min Abso- Type of μg/ C_(Max)T_(Max) / lute Example Insulin Route animal μU/ml min ml BA  9 (P-Insulin IT 80 56 15 12878 2001- 25)  9 750-1 IT 100 64 368 27954 PEG  9750-1 IT 300 160 188 50691 PEG  9 750-1 IT 500 3474 184 255881 PEG 10(P- Insulin IT 80 132 15 28167 2002- 001) 10 750-1 IT 80 56 210 36818PEG 10 750-1 IT 160 117 78 60713 PEG IV Ref. Insulin IV 20 3057 5 44388P-2002- 002 IV Ref. 750-2 IV 20 2638 7 63190 PEG IV Ref. 750-2 IV 303510 5 62746 PEG 11 (P- insulin IT 80 89 24 22203 12.5 2002- 003) 11750-2 IT 80 164 73 57639 32** PEG 32***

[0239] Absolute bioavailability was calculated as follows:$\frac{({AUC})}{\left( {AUC}_{{IV}\quad {ins}} \right)}\frac{\left( {Dose}_{{IV}\quad {ins}} \right)}{({Dose})}$

Example 12 Administration of 2K PEG Insulin to the Lung (P-2002-010)

[0240] Another exemplary pegylated insulin composition, 2K PEG insulin,was administered to rats by intratracheal administration. The 2K PEGinsulin used for this study was prepared as described in Example 3.Animals were dosed at 80 μg insulin per animal for non-pegylatedinsulin. Animals were dosed at 300 μg insulin per animal, 600 μg insulinper animal, 900 μg insulin per animal, and 1200 μg insulin per animalfor 2K PEG insulin. The in-vivo rat study was conducted essentially asdescribed in Example 8 above. The precise dosing regimen followed anddoses administered are summarized in the tables below. TABLE 14 TotalRoute of Number of Daily Dose No of Type of Adminis- Animals/ of InsulinDosing Group No. Insulin tration Gender (μg/animal) Days 1 Insulin i.t.3 80 1 2 PEG2K-1 i.t. 3 600 1 Insulin 3 PEG2K-1 i.t. 3 80 1 Insulin 4PEG2K-1 i.t. 3 160 1 Insulin 5 PEG2K-1 i.t. 3 300 1 Insulin 6 PEG2K-1i.t. 3 900 1 Insulin 7 PEG2K-1 i.t. 3 1200 1 Insulin

[0241] TABLE 15 Total Daily Dose Dose Concentration of Group Type of ofInsulin Volume Dosing No. Insulin (μg/animal) (μl) Solution (mg/ml) 1Insulin 80 300 0.267 2 PEG2K-1 600 300 2.0 Insulin 3 PEG2K-1 80 3000.267 Insulin 4 PEG2K-1 160 300 0.533 Insulin 5 PEG2K-1 300 300 1.0Insulin 6 PEG2K-1 900 300 3.0 Insulin 7 PEG2K-1 1200 300 4.0 Insulin

[0242] A plot of mean blood glucose concentrations followingintratracheal administration is shown in FIG. 12. Good dose response wasobserved for the pegylated insulin compositions when administered to thelung (i.e., higher doses of 2K PEG insulin resulted in a greaterdecrease in blood glucose concentration). Although the time point in thecurve at which maximum glucose suppression was achieved appears to beabout 3 hours for both the pegylated and non-pegylated compositions, theprofiles for the pegylated versus non-pegylated insulin differsignificantly with respect to duration of glucose suppression. Inparticular, for time points past about 6 hours, at the three higher 2Kpegylated insulin doses (600 μg, 900 μg, and 1200 μg per animal),glucose levels were suppressed significantly below those ofnon-pegylated insulin. These results further demonstrate that aprolonged systemic effect can be achieved by administration of pegylatedinsulin to the lung.

Example 13 Evaluation of Serum Glucose and Insulin ConcentrationsFollowing Intravenous Administration of 2K PEG Insulin in Rats(P-2002-009)

[0243] This study was conducted to further explore the activity ofinsulin in an exemplary 2K PEG insulin composition, and to determine theintravenous (i.v.) dose of pegylated human insulin (PEG2K-1) effectiveto lower blood glucose to a concentration of about 30 -40 mg/dL.

[0244] A protocol similar to that described in Example 7 was conductedusing the compositions, animal groups, and doses summarized in thetables below. TABLE 16 Total Route of Number of Daily Dose No of Type ofAdminis- Animals/ of Insulin Dosing Group No. Insulin tration Gender(μg/animal) Days 1 Insulin i.v. 2M 20 1 2 PEG2K-1 i.v. 2M 20 1 Insulin 3PEG2K-1 i.v. 2M 30 1 Insulin 4 PEG2K-1 i.v. 2M 40 1 Insulin 5 PEG2K-1i.v. 2M 80 1 Insulin 6 PEG2K-1 i.v. 2M 160 1 Insulin

[0245] TABLE 17 Control/ Total Daily Dose Dose Concentration Test ofInsulin Volume of Dosing Group No. Article (μg/animal) (μl) Solution(□g/ml) 1 Insulin 20 300 67 2 PEG2 20 300 67 K-1 Insulin 3 PEG2 30 300100 K-1 Insulin 4 PEG2 40 300 133 K-1 Insulin 5 PEG2 80 300 267 K-1Insulin 6 PEG2 160 300 533 K-1 Insulin

[0246] A plot of mean serum insulin concentrations following intravenousadministration of non-pegylated and 2K PEG insulin at doses of 20μg/animal (non-pegylated insulin) and 20, 30 and 40 μg/animal (2K PEGinsulin) is shown in FIG. 13. A plot of mean blood glucoseconcentrations following intravenous administration of non-pegylated and2K PEG insulin at the doses described above is shown in FIG. 14.

It is claimed:
 1. An insulin composition for pulmonary administration,said composition comprising a conjugate of insulin covalently coupled toone or more molecules of a non-naturally occurring hydrophilic polymer.2. The insulin composition of claim 1, wherein said conjugate is absenta lipophilic moiety.
 3. The insulin composition of claim 1, wherein saidnon-naturally occurring hydrophilic polymer is a polyalkyene glycol. 4.The insulin composition of claim 1, wherein said non-naturally occurringhydrophilic polymer is polyethylene glycol.
 5. The insulin compositionof claim 2, wherein said non-naturally occurring hydrophilic polymer ispolyethylene glycol.
 6. The composition of claim 4, characterized by anabsolute pulmonary bioavailability that is greater than that of nativeinsulin.
 7. The composition of claim 6, characterized by an absolutepulmonary bioavailability that is at least twice that of native insulin.8. The composition of claim 4, characterized by an absolute pulmonarybioavailability greater than 15%.
 9. The composition of claim 8,characterized by an absolute pulmonary bioavailability greater than 30%.10. The composition of claim 4, which when administered to the lung, ischaracterized by a Tmax that is at least three times that of nativeinsulin.
 11. The composition of claim 10, which when administered to thelung, is characterized by a Tmax that is at least five times that ofnative insulin.
 12. The composition of claim 4, wherein saidpolyethylene glycol is end-capped.
 13. The composition of claim 12,wherein said polyethylene glycol is end-capped with an alkoxy group. 14.The composition of claim 4, wherein said polyethylene glycol is selectedfrom the group consisting of linear polyethylene glycol, branchedpolyethylene glycol, forked polyethylene glycol, and dumbbellpolyethylene glycol.
 15. The composition of claim 14, wherein saidpolyethylene glycol comprises a biodegradable linkage.
 16. Thecomposition of claim 14, wherein said polyethylene glycol comprises anumber of (OCH₂CH₂) subunits selected from the group consisting of fromabout 2 to 300 subunits, from about 4 to 200 subunits, and from about 10to 100 subunits.
 17. The composition of claim 14, wherein saidpolyethylene glycol has a nominal average molecular weight from about200 to about 10,000 daltons.
 18. The composition of claim 14, whereinsaid polyethylene glycol is linear.
 19. The composition of claim 17,wherein said polyethylene glycol has a nominal average molecular weightfrom about 200 to about 5,000 daltons.
 20. The composition of claim 17,wherein said polyethylene glycol has a nominal average molecular weightfrom about 200 to about 2,000 daltons.
 21. The composition of claim 17,wherein said polyethylene glycol has a nominal average molecular weightfrom about 200 to about 1,000 daltons.
 22. The composition of claim 4,wherein said insulin is native insulin.
 23. The composition of claim 4,wherein said conjugate has a purity of greater than 90%.
 24. Thecomposition of claim 4, wherein said insulin is covalently coupled topolyethylene glycol at one or more of its amino sites.
 25. Thecomposition of claim 24, wherein at least about 75% of the B-1Phe siteson insulin are covalently coupled to polyethylene glycol.
 26. Thecomposition of claim 25, wherein at least about 90% of the B-1Phe siteson insulin are covalently coupled to polyethylene glycol.
 27. Thecomposition of claim 24, comprising a mixture of monomer and dimerconjugates of insulin.
 28. The composition of claim 27, furthercomprising a trimer insulin conjugate.
 29. The composition of claim 4,wherein said insulin is covalently coupled to polyethylene glycol via alinking moiety positioned at a terminus of said polyethylene glycol. 30.The composition of claim 4, wherein said polyethylene glycol, prior tocoupling with insulin, possesses an activated linking moiety at oneterminus suitable for covalent coupling with insulin.
 31. Thecomposition of claim 30, wherein said activated linking moiety issuitable for coupling with reactive insulin amino groups.
 32. Thecomposition of claim 31, wherein said activated linking moiety comprisesa reactive functional group selected from the group consisting ofN-hydroxysuccinimide active esters, active carbonates, aldehydes, andacetals.
 33. The composition of claim 29, wherein insulin is covalentlycoupled to polyethylene glycol via an amide linkage.
 34. The compositionof claim 4 in aerosolized form.
 35. The composition of claim 4 in liquidor dry form.
 36. The composition of claim 4 further comprising apharmaceutically acceptable excipient.
 37. The composition of claim 4 inspray-dried form.
 38. A method for delivering insulin to a mammaliansubject in need thereof, said method comprising: aerosolizing theinsulin composition of claim 4, and administering said aerosolizedinsulin composition by inhalation for deposition in and absorption fromthe lung of said subject.
 39. A method for providing a substantiallynon-immunogenic insulin composition for administration to the lung of asubject in need thereof, said method comprising: covalently couplinginsulin to one or more molecules of a non-naturally occurringhydrophilic polymer to provide a composition comprising aninsulin-hydrophilic polymer conjugate, and administering saidcomposition to the lung of a subject in need thereof by inhalation,whereby as a result of said administering, said insulin passes throughthe lung and enters into the blood circulation.
 40. The method of claim39, wherein said non-naturally occurring hydrophilic polymer is apolyalkylene glycol.
 41. A method for providing a prolonged-effectinsulin composition for administration to the lung of a subject in needthereof, said method comprising: covalently coupling insulin to one ormore molecules of a non-naturally occurring hydrophilic polymer toprovide a composition comprising an insulin-hydrophilic polymerconjugate, administering said composition to the lung of a subject inneed thereof by inhalation, whereby as a result of said administering,(i) said insulin passes through the lung and enters the bloodcirculation, and (ii) elevated blood levels of insulin are sustained forat least 8 hours post administration.
 42. The method of claim 41,wherein said non-naturally occurring hydrophilic polymer is apolyalkylene glycol.
 43. The method of claim 42, wherein saidnon-naturally occurring hydrophilic polymer is polyethylene glycol. 44.The method of claim 43, whereby elevated levels of insulin are sustainedfor at least 10 hours post-administration.
 45. The method of claim 43,whereby elevated levels of insulin are sustained for at least 12 hourspost-administration.
 46. The method of claim 43, whereby further as aresult of said administering, glucose levels in said subject aresuppressed for at least 10 hours post administration.
 47. The method ofclaim 46, whereby further as a result of said administering, glucoselevels in said subject are suppressed for at least 12 hours postadministration.
 48. The method of claim 43, wherein said administeringstep comprises administering said composition in aerosolized form. 49.The method of claim 43, further comprising the step of aerosolizing saidcomposition prior to administering.
 50. The method of claim 43, whereinsaid coupling step comprises covalently coupling insulin to polyethyleneglycol in a site-specific fashion.
 51. The method of claim 43, whereinsaid coupling step comprises covalently coupling insulin to polyethyleneglycol in a random fashion.
 52. The method of claim 43, wherein saidconjugate when administered to the lung is further characterized by anabsolute pulmonary bioavailability that is greater than that of nativeinsulin.
 53. The method of claim 43, wherein said coupling stepcomprises covalently coupling insulin to one or more molecules ofend-capped polyethylene glycol.
 54. The method of claim 43, wherein saidcoupling step comprises covalently coupling insulin to one or moremolecules of polyethylene glycol selected from the group consisting oflinear, branched, forked, and dumbbell polyethylene glycol.
 55. Themethod of claim 43, wherein said conjugate is absent a lipophilicmoiety.
 56. The method of claim 43, wherein said composition is absent alipophilic component.
 57. The method of claim 43, wherein said couplingstep comprises covalently coupling insulin to one or more molecules ofpolyethylene glycol comprising a biodegradable linkage.
 58. The methodof claim 43, wherein said polyethylene glycol comprises a number of(OCH₂CH₂) subunits selected from the group consisting of from about 2 to300 subunits, from about 4 to 200 subunits, and from about 10 to 100subunits.
 59. The method of claim 43, wherein said polyethylene glycolhas a nominal average molecular weight from about 200 to about 10,000daltons.
 60. The method of claim 43, wherein said polyethylene glycolhas a nominal average molecular weight from about 200 to about 5,000daltons.
 61. The method of claim 43, wherein said polyethylene glycolhas a nominal average molecular weight from about 200 to about 2,000daltons.
 62. The method of claim 43, wherein said polyethylene glycolhas a nominal average molecular weight from about 200 to about 1,000daltons.
 63. The method of claim 43, wherein said coupling comprisescoupling polyethylene glycol to insulin at one or more of its reactiveamino sites.
 64. The method of claim 63, wherein said polyethyleneglycol is coupled to insulin at more or more of its reactive amino sitesvia a bond selected from the group consisting of amide, urethane, andmethylene amino.
 65. The method of claim 63, wherein said couplingcomprises reacting a polyethylene glycol having a terminal reactivegroup selected from the group consisting of N-hydroxysuccinimide activeesters, active carbonates, aldehydes, and acetals with one or morereactive amino sites on insulin.
 66. The method of claim 43, whereinsaid coupling results in a composition wherein at least about 75% of theB-1Phe sites on insulin are covalently coupled to polyethylene glycol.67. The method of claim 43, wherein said coupling results in acomposition wherein at least about 90% of the B-1Phe sites on insulinare covalently coupled to polyethylene glycol.
 68. The method of claim43, wherein said coupling results in a composition comprising a mixtureof monomer and dimer conjugates of insulin.
 69. The method of claim 68,wherein said coupling results in a composition further comprising atrimer conjugate of insulin.
 70. The method of claim 43, wherein saidpolyethylene glycol comprises an activated linking moiety at oneterminus suitable for covalent coupling with insulin.
 71. The method ofclaim 43, wherein said activated linking moiety comprises a reactivefunctional group selected from the group consisting ofN-hydroxysuccinimide active esters, active carbonates, aldehydes, andacetals.
 72. The method of claim 70, wherein said linking moiety has alength of from about 2 to about 20 atoms.
 73. The method of claim 43,wherein said administering step comprises administering said compositionby dry powder inhaler.
 74. The method of claim 43, wherein saidadministering step comprises administering said composition by a metereddose inhaler.
 75. The method of claim 43, wherein said administeringstep comprises administering said composition by a nebulizer.
 76. Themethod of claim 43, wherein said composition further comprises apharmaceutically acceptable excipient.
 77. The method of claim 43,whereby as a result of administering said conjugate composition, serumlevels of insulin that are at least 2 times greater than basal levelsare achieved within 1 hour post administration.